Human IL-23 antigen binding proteins

ABSTRACT

Antigen binding proteins that bind to human IL-23 protein are provided. Nucleic acids encoding the antigen binding protein, vectors, and cells encoding the same as well as use of IL-23 antigen binding proteins for diagnostic and therapeutic purposes are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 14/228,556 filed Mar. 28, 2014 (now U.S. Pat. No. 9,487,580)which is a continuation of U.S. patent application Ser. No. 13/504,449filed Aug. 31, 2012 (now U.S. Pat. No. 8,722,033), which in turn is aUnited States National Stage Application of PCT/US10/54148, filed Oct.26, 2010, which in turn claims the benefit under 35 U.S.C. 119(e) ofU.S. Provisional Application No. 61/254,982, filed Oct. 26, 2009 andU.S. Provisional Application No. 61/381,287, filed Sep. 9, 2010, whichare incorporated herein by reference.

BACKGROUND

Interleukin 23 (IL-23), a heterodimeric cytokine, is a potent inducer ofpro-inflammatory cytokines. IL-23 is related to the heterodimericcytokine Interleukin 12 (IL-12) both sharing a common p40 subunit. InIL-23, a unique p19 subunit is covalently bound to the p40 subunit. InIL-12, the unique subunit is p35 (Oppmann et al., Immunity, 2000, 13:713-715). The IL-23 heterodimeric protein is secreted. Like IL-12, IL-23is expressed by antigen presenting cells (such as dendritic cells andmacrophages) in response to activation stimuli such as CD40 ligation,Toll-like receptor agonists and pathogens. IL-23 binds a heterodimericreceptor comprising an IL-12Rβ1 subunit (which is shared with the IL-12receptor) and a unique receptor subunit, IL-23R. The IL-12 receptorconsists of IL-12Rβ1 and IL-12Rβ2. IL-23 binds its heterodimericreceptor and signals through JAK2 and Tyk2 to activate STAT1, 3, 4 and 5(Parham et al., J. Immunol. 2002, 168:5699-708). The subunits of thereceptor are predominantly co-expressed on activated or memory T cellsand natural killer cells and also at lower levels on dendritic cells,monocytes, macrophages, microglia, keratinocytes and synovialfibroblasts. IL-23 and IL-12 act on different T cell subsets and playsubstantially different roles in vivo.

IL-23 acts on activated and memory T cells and promotes survival andexpansion of the T cell subset, Th17. Th17 cells produce proinflammatorycytokines including IL-6, IL-17, TNFα, IL-22 and GM-CSF. IL-23 also actson natural killer cells, dendritic cells and macrophages to inducepro-inflammatory cytokine expression. Unlike IL-23, IL-12 induces thedifferentiation of naïve CD4+ T cells into mature Th1 IFNγ-producingeffector cells, and induces NK and cytotoxic T cell function bystimulating IFNγ production. Th1 cells driven by IL-12 were previouslythought to be the pathogenic T cell subset in many autoimmune diseases,however, more recent animal studies in models of inflammatory boweldisease, psoriasis, inflammatory arthritis and multiple sclerosis, inwhich the individual contributions of IL-12 versus IL-23 were evaluatedhave firmly established that IL-23, not IL-12, is the key driver inautoimmune/inflammatory disease (Ahern et al., Immun. Rev. 2008226:147-159; Cua et al., Nature 2003 421:744-748; Yago et al., ArthritisRes and Ther. 2007 9(5): R96). It is believed that IL-12 plays acritical role in the development of protective innate and adaptiveimmune responses to many intracellular pathogens and viruses and intumor immune surveillance. See Kastelein, et al., Annual Review ofImmunology, 2007, 25: 221-42; Liu, et al., Rheumatology, 2007, 46(8):1266-73; Bowman et al., Current Opinion in Infectious Diseases, 200619:245-52; Fieschi and Casanova, Eur. J. Immunol. 2003 33:1461-4; Meeranet al., Mol. Cancer Ther. 2006 5: 825-32; Langowski et al., Nature 2006442: 461-5. As such, IL-23 specific inhibition (sparing IL-12 or theshared p40 subunit) should have a potentially superior safety profilecompared to dual inhibition of IL-12 and IL-23.

Therefore, use of IL-23 specific antagonists that inhibit human IL-23(such as antibodies that bind at least the unique p19 subunit or bindboth the p19 and p40 subunits of IL-23) that spare IL-12 should provideefficacy equal to or greater than IL-12 antagonists or p40 antagonistswithout the potential risks associated with inhibition of IL-12. Murine,humanized and phage display antibodies selected for inhibition ofrecombinant IL-23 have been described; see for example U.S. Pat. No.7,491,391, WIPO Publications WO1999/05280, WO2007/0244846,WO2007/027714, WO 2007/076524, WO2007/147019, WO2008/103473, WO2008/103432, WO2009/043933 and WO2009/082624. However, there is a needfor fully human therapeutic agents that are able to inhibit native humanIL-23. Such therapeutics are highly specific for the target,particularly in vivo. Complete inhibition of the in vivo target canresult in lower dose formulations, less frequent and/or more effectivedosing which in turn results in reduced cost and increased efficiency.The present invention provides such IL-23 antagonists.

SUMMARY

Antigen binding proteins that bind IL-23, particularly native humanIL-23, are provided. The human IL-23 antigen binding proteins canreduce, inhibit, interfere with, and/or modulate at least one of thebiological responses related to IL-23, and as such, are useful forameliorating the effects of IL-23 related diseases or disorders. IL-23antigen binding proteins can be used, for example, to reduce, inhibit,interfere with and/or modulate IL-23 signaling, IL-23 activation of Th17cells, IL-23 activation of NK cells, or inducing production ofproinflammatory cytokines.

Also provided are expression systems, including cell lines, for theproduction of IL-23 antigen binding proteins and methods of diagnosingand treating diseases related to human IL-23.

Some of the antigen binding proteins that bind IL-23 that are providedcomprise at least one heavy chain variable region comprising a CDRH1, aCDRH2 and a CDRH3 selected from the group consisting of: a CDRH1 thatdiffers by no more than one amino acid substitution, insertion ordeletion from a CDRH1 as shown in TABLE 3; a CDRH2 that differs by nomore than three, two or one amino acid substitutions, insertions and/ordeletions from a CDRH2 as shown in TABLE 3; a CDRH3 that differs by nomore than three, two or one amino acid substitutions, insertions and/ordeletions from a CDRH3 as shown in TABLE 3; and comprising at least onelight chain variable region comprising a CDRL1, a CDRL2 and a CDRL3selected from the group consisting of: a CDRL1 that differs by no morethan three, two or one amino acid substitutions, insertions and/ordeletions from a CDRL1 as shown in TABLE 3; a CDRL2 that differs by nomore than one amino acid substitution, insertion or deletion from aCDRL2 as shown in TABLE 3; a CDRL3 that differs by no more than oneamino acid substitution, insertion or deletion from a CDRL3 as shown inTABLE 3. In one embodiment is provided isolated antigen binding proteinscomprising: a CDRH1 selected from the group consisting of SEQ ID NO: 91,94, 97, 100, and 103; a CDRH2 selected from the group consisting of SEQID NO:92, 95, 98, 101, 104, 107, and 110; a CDRH3 selected from thegroup consisting of SEQ ID NO: 93, 96, 99, 102, and 105; a CDRL1selected from the group consisting of SEQ ID NO: 62, 65, 68, 71, and 74;a CDRL2 selected from the group consisting of SEQ ID NO:63, 66, 69, 72,75, and 78; and a CDRL3 selected from the group consisting of SEQ IDNO:64, 67, 70 and 73. In another embodiment is provided isolated antigenbindings protein of comprising: a CDRH1 selected from the groupconsisting of SEQ ID NO: 91, 106, 109, 112, and 115; a CDRH2 selectedfrom the group consisting of SEQ ID NO: 113, 116, 118, 120, 121, and122; a CDRH3 selected from the group consisting of SEQ ID NO: 108, 111,114, 117, and 119; a CDRL1 selected from the group consisting of SEQ IDNO: 77, 80, 83, 85, 86, 87, 88, 89 and 90; a CDRL2 is SEQ ID NO: 81; anda CDRL3 selected from the group consisting of SEQ ID NO: 76, 79, 82 and84. In another embodiment is provided an isolated antigen-bindingprotein of that comprises at least one heavy chain variable region andat least one light chain variable region. In yet another embodiment isprovided an isolated antigen-binding protein as described above thatcomprise at least two heavy chain variable regions and at least twolight chain variable regions. In yet another embodiment is provided anisolated antigen binding protein wherein the antigen binding protein iscoupled to a labeling group.

Also provided are isolated antigen binding proteins that bind IL-23selected from the group consisting of a) an antigen binding proteinhaving CDRH1 of SEQ ID NO:129, CDRH2 of SEQ ID NO:132, CDRH3 of SEQ IDNO:136, and CDRL1 of SEQ ID NO:123, CDRL2 of SEQ ID NO:81, and CDRL3 ofSEQ ID NO: 76; b) an antigen binding protein having CDRH1 of SEQ IDNO:131, CDRH2 of SEQ ID NO: 134, CDRH3 of SEQ ID NO:137 and CDRL1 of SEQID NO:124, CDRL2 of SEQ ID NO126 and CDRL3 of SEQ ID NO:128; c) a) anantigen binding protein having CDRH1 of SEQ ID NO:130, CDRH2 of SEQ IDNO:133, CDRH3 of SEQ ID NO:99 and CDRL1 of SEQ ID NO:68, CDRL2 of SEQ IDNO:69, and CDRL3 of SEQ ID NO:67; and d) an antigen binding proteinhaving CDRH1 SEQ ID NO:91, CDRH2 SEQ ID NO: 135, CDRH3 SEQ ID NO:138 andCDRL1 SEQ ID NO:125, CDRL2 SEQ ID NO:127, and CDRL3 SEQ ID NO:64.

Also provided are isolated antigen binding proteins that bind IL-23comprising at least one heavy chain variable region and at least onelight chain variable region, selected from the group consisting of: aheavy chain variable region comprising amino acid residues 31-35, 50-65and 99-113 of SEQ ID NO:31; and a light chain variable region comprisingamino acid residues 23-36, 52-58 and 91-101 of SEQ ID NO:1; a heavychain variable region comprising amino acid residues 31-35, 50-65 and99-110 of SEQ ID NO:34 and heavy chain variable region comprising aminoacid residues 31-35, 50-66 and 99-110 of SEQ ID NO:36; and a light chainvariable region comprising amino acid residues 23-36, 52-62 and 97-105of SEQ ID NO:4; a heavy chain variable region comprising amino acidresidues 31-35, 50-66 and 99-114 of SEQ ID NO:38; and a light chainvariable region comprising amino acid residues 23-34, 50-61 and 94-106of SEQ ID NO:7; a heavy chain variable region comprising amino acidresidues 31-35, 50-66 and 99-114 of SEQ ID NO:40; and a light chainvariable region comprising amino acid residues 24-34, 50-56 and 94-106of SEQ ID NO:9; a heavy chain variable region comprising amino acidresidues 31-35, 50-66 and 99-114 of SEQ ID NO:42; and a light chainvariable region comprising amino acid residues 23-34, 50-61 and 94-106of SEQ ID NO:11; a heavy chain variable region comprising amino acidresidues 31-35, 50-65 and 98-107 of SEQ ID NO:44; and a light chainvariable region comprising amino acid residues 24-34, 50-56 and 89-97 ofSEQ ID NO:13; a heavy chain variable region comprising amino acidresidues 31-37, 52-67 and 100-109 of SEQ ID NO:46 or SEQ ID NO:153; anda light chain variable region comprising amino acid residues 24-34,50-56 and 89-97 of SEQ ID NO15; a heavy chain variable region comprisingamino acid residues 31-37, 52-67 and 100-109 of SEQ ID NO:48; and alight chain variable region comprising amino acid residues 24-34, 50-56and 89-97 of SEQ ID NO:17; a heavy chain variable region comprisingamino acid residues 31-37, 52-67 and 101-109 of SEQ ID NO:50; and alight chain variable region comprising amino acid residues 24-34, 50-56and 89-97 of SEQ ID NO:19; a heavy chain variable region comprisingamino acid residues 31-35, 50-65 and 98-107 of SEQ ID NO: 52; and alight chain variable region comprising amino acid residues 24-34, 50-56and 98-107 of SEQ ID NO:21; a heavy chain variable region comprisingamino acid residues 31-37, 52-67 and 100-109 of SEQ ID NO:54; and alight chain variable region comprising amino acid residues 24-34, 50-56and 89-97 of SEQ ID NO:23; a heavy chain variable region comprisingamino acid residues 31-37, 52-67 and 100-109 of SEQ ID NO:56; and alight chain variable region comprising amino acid residues 24-34, 50-56and 89-97 of SEQ ID NO:25; and a heavy chain variable region comprisingamino acid residues 31-37, 52-57 and 100-109 of SEQ ID NO:58; and alight chain variable region comprising amino acid residues 24-34, 500-56and 89-97 of SEQ ID NO:27.

Provided herein is an isolated antigen binding protein that binds IL-23comprising a heavy chain variable region and a light chain variableregion, wherein the heavy chain variable region sequence differs by nomore than 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acidsubstitutions, additions and/or deletions from a heavy chain variableregion sequence as shown in TABLE 2; and wherein the light chainvariable region sequence differs by no more than 13, 12, 11, 10, 9, 8,7, 6, 5, 4, 3, 2 or 1 amino acid substitutions, additions and/ordeletions from a light chain variable region sequence as shown in TABLE1.

Also provided is an isolated antigen binding protein that binds IL-23selected from the group consisting of a) a heavy chain variable regionof SEQ ID NO:140 and a light chain variable region of SEQ ID NO: 30; b)a heavy chain variable region of SEQ ID NO:141 and a light chainvariable region of SEQ ID NO:61; c) a heavy chain variable region of SEQID NO:142 and a light chain variable region of SEQ ID NO:4; and d) aheavy chain variable region of SEQ ID NO:143 and a light chain variableregion of SEQ ID NO:139.

Also provided is an isolated antigen binding protein comprising a heavychain variable region comprising of an amino acid sequence having atleast 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:31,34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56 and 58; and a light chainvariable region comprising an amino acid sequence having at least 90%sequence identity to SEQ ID NO: 1, 4, 7, 9, 11, 13, 15, 17, 19, 21, 23,25 and 27. In another embodiment is an isolated antigen binding proteincomprising a heavy chain variable region selected from the groupconsisting of SEQ ID NO: 44, 46, 48, 50, 52, 54, 56, 58 and 153, and alight chain variable region selected from the group consisting of SEQ IDNO:13, 15, 17, 19, 21, 23, 25, and 27. In yet another embodiment is anisolated antigen binding protein comprising a heavy chain variableregion selected from the group consisting of SEQ ID NO: 31, 34, 36, 38,40 and 42, and a light chain variable region selected from the groupconsisting of SEQ ID NO: 1, 4, 7, 9 and 11.

Also provided is an isolated antigen binding protein that binds IL-23comprising a heavy chain variable region and a light chain variableregion selected from the group consisting of: a) a heavy chain variableregion of SEQ ID NO:31 and a light chain variable region of SEQ ID NO:1;b) a heavy chain variable region of SEQ ID NO:34 or 36 and a light chainvariable region of SEQ ID NO:4; c) a heavy chain variable region of SEQID NO:38 and a light chain variable region of SEQ ID NO: 7; d) a heavychain variable region of SEQ ID NO:40 and a light chain variable regionof SEQ ID NO:9; e) a heavy chain variable region of SEQ ID NO:42 and alight chain variable region of SEQ ID NO: 11; f) a heavy chain variableregion of SEQ ID NO:44 and a light chain variable region of SEQ IDNO:13; g) a heavy chain variable region of SEQ ID NO:46 or SEQ ID NO:153and a light chain variable region of SEQ ID NO:15; h) a heavy chainvariable region of SEQ ID NO:48 and a light chain variable region of SEQID NO:17; i) a heavy chain variable region of SEQ ID NO:50 and a lightchain variable region of SEQ ID NO: 19; j) a heavy chain variable regionof SEQ ID NO:52 and a light chain variable region of SEQ ID NO:21; k) aheavy chain variable region of SEQ ID NO:54 and a light chain variableregion of SEQ ID NO:23; l) a heavy chain variable region of SEQ ID NO:56and a light chain variable region of SEQ ID NO:25; and m) a heavy chainvariable region of SEQ ID NO:58 and a light chain variable region of SEQID NO:27.

Also provided is an isolated antigen binding protein that binds humanIL-23, wherein the covered patch formed when the antigen binding proteinis bound to human IL-23 comprises residue contacts 30, 31, 32, 49, 50,52, 53, 56, 92 and 94 of SEQ ID NO:15, wherein the residue contacts havea difference value of greater than or equal to 10 Å² as determined bysolvent exposed surface area. Within one embodiment the residue contactscomprise residues 31-35, 54, 58-60, 66, and 101-105 of SEQ ID NO:46.

Also provided is an isolated antigen binding protein that binds humanIL-23, wherein the covered patch formed when the antigen binding proteinis bound to human IL-23 comprises residue contacts 31-34, 51, 52, 55,68, 93 and 98 of SEQ ID NO:1, wherein the residue contacts have adifference value of greater than or equal to 10 Å² as determined bysolvent exposed surface area. Within one embodiment the residue contactscomprise residues 1, 26, 28, 31, 32, 52, 53, 59, 76, 101, 102 and104-108 of SEQ ID NO:31.

Also provided is an isolated antigen binding protein that binds humanIL-23, wherein when the antigen binding protein is bound to human IL-23,the antigen binding protein is 5 Å or less from residues 32-35, 54,58-60, 66 and 101-105 of SEQ ID NO:46, as determined by X-raycrystallography. In one embodiment the antigen binding protein is 5 Å orless from residues 31-35, 54, 56, 58-60, 66 and 101-105 of SEQ ID NO:46.

Also provided is an isolated antigen binding protein that binds humanIL-23, wherein when the antigen binding protein is bound to human IL-23,the antigen binding protein is 5 Å or less from residues 30-32, 49, 52,53, 91-94 and 96 of SEQ ID NO:15, as determined by X-raycrystallography. In one embodiment the antigen binding protein is 5 Å orless from residues 30-32, 49, 50, 52, 53, 56, 91-94 and 96 of SEQ IDNO:15.

Also provided is an isolated antigen binding protein that binds humanIL-23, wherein when the antigen binding protein is bound to human IL-23,the antigen binding protein is 5 Å or less from residues 26-28, 31, 53,59, 102 and 104-108 of SEQ ID NO:31, as determined by X-raycrystallography. In one embodiment the antigen binding protein is 5 Å orless from residues 1, 26-28, 30-32, 52, 53, 59, 100, and 102-108 of SEQID NO:31.

Also provided is an isolated antigen binding protein that binds humanIL-23, wherein when said antigen binding protein is bound to humanIL-23, said antigen binding protein is 5 Å or less from residues 31-34,51, 52, 55, 68 and 93 of SEQ ID NO:1 as determined by X-raycrystallography. In one embodiment the antigen binding protein is 5 Å orless from residues 29, 31-34, 51, 52, 55, 68, 93 and 100 of SEQ ID NO:1.

Also provided is an isolated antigen binding protein as described above,wherein the antigen binding protein is an antibody. In one embodiment isprovided an isolated antigen binding protein wherein the antibody is amonoclonal antibody, a recombinant antibody, a human antibody, ahumanized antibody, a chimeric antibody, a multispecific antibody, or anantibody fragment thereof. In another embodiment is provided an isolatedantigen binding protein wherein the antibody fragment is a Fab fragment,a Fab′ fragment, a F(ab′)2 fragment, a Fv fragment, a diabody, or asingle chain antibody molecule. In yet another embodiment is provided anisolated antigen binding protein wherein the antigen binding protein isa human antibody. In still another embodiment is provided an isolatedantigen binding protein wherein the antigen binding protein is amonoclonal antibody. In another embodiment is provided an isolatedantigen binding protein wherein the antigen binding protein is of theIgG1-, IgG2-IgG3- or IgG4-type. In yet another embodiment is provided anisolated antigen binding protein wherein the antigen binding protein isof the IgG1- or IgG2-type.

An isolated nucleic acid molecule encoding an antigen binding protein asdescribed above, is also provided. In one embodiment is provided anisolated nucleic acid molecule wherein at least one heavy chain variableregion is encoded by an isolated nucleic acid molecule selected from thegroup consisting of SEQ ID NOs:32, 35, 37, 39, 41, 43, 45, 47, 49, 51,53, 55, 57, 59 and 152 and at least one light chain variable region isencoded by an isolated nucleic acid molecule selected from the groupconsisting of SEQ ID NOs:2, 5, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26,and 28. In another embodiment is provided a nucleic acid moleculewherein the nucleic acid molecule is operably linked to a controlsequence. In another embodiment is provided a vector comprising anucleic acid molecule as described above. In yet another embodiment isprovided a host cell comprising the nucleic acid molecule as describedabove. In another embodiment is provided a host cell comprising thevector described above. In yet another embodiment is provided anisolated polynucleotide sufficient for use as a hybridization probe, PCRprimer or sequencing primer that is a fragment of the nucleic acidmolecule as described above or its complement.

Also provided is a method of making the antigen binding protein asdescribed above, comprising the step of preparing said antigen bindingprotein from a host cell that secretes said antigen binding protein.

Also provided is an isolated antigen binding protein that binds humanIL-23, wherein the covered patch formed when the antigen binding proteinis bound to human IL-23 comprises a residue contact within residues46-58, a residue contact within residues 112-120, and a residue contactwithin residues 155-163 of the human IL-23p19 subunit as described inSEQ ID NO:145, wherein the residue contact has a difference valuegreater than or equal to 10 Å² as determined by solvent exposed surfacearea. In one embodiment is provided wherein the covered patch formedwhen the antigen binding protein is bound to human IL-23 comprises one,two, three, four, five, six, seven, eight, nine, ten, eleven, twelve orthirteen residue contacts within residues 46-58, one, two, three, four,five, six, seven, eight, nine or ten residue contacts within residues112-120, and one, two, three, four, five, six, seven, eight or nineresidue contacts within residues 155-163 of the human IL-23p19 subunitas described in SEQ ID NO:145. In another embodiment is provided whereinthe covered patch formed when the antigen binding protein binds to humanIL-23 comprises a residue contact within residues 121-125 of the humanIL-23p40 subunit as described in SEQ ID NO:147. In a related embodimentis wherein the covered patch formed when the antigen binding protein isbound to human IL-23 comprises one, two, three, four or five residuecontacts within residues 121-125 of the human IL-23p40 subunit asdescribed in SEQ ID NO:147. Within another embodiment is providedwherein the covered patch formed when the antigen binding protein isbound to human IL-23 comprises residue contacts 46, 47, 49, 50, 53,112-116, 118, 120, 155, 156, 159, 160, and 163 of SEQ ID NO:145. Inanother embodiment is provided wherein the covered patch formed when theantigen binding protein is bound to human IL-23 comprises residuecontacts 46, 47, 49, 50, 53, 112-118, 120, 155, 156, 159, 160, and 163of SEQ ID NO:145. Within another embodiment is provided wherein thecovered patch formed when the antigen binding protein is bound to humanIL-23 comprises residues 46, 47, 49, 50, 53-55, 57, 58, 112-116,118-120, 155, 156, 159, 160, 162 and 163 of SEQ ID NO:145. In a relatedembodiment is provided wherein the covered patch formed when the antigenbinding protein is bound to human IL-23 comprises residue contact 122 ofthe human IL-23p40 subunit as described in SEQ ID NO:147. In anotherrelated embodiment is provided wherein the covered patch formed when theantigen binding protein is bound to human IL-23 comprises residuecontacts 122 and 124 of the human IL-23p40 subunit as described in SEQID NO:147. In yet another related embodiment is provided wherein thecovered patch formed when the antigen binding protein is bound to humanIL-23 comprises residue contact 121-123 and 125 of the human IL-23p40subunit as described in SEQ ID NO:147. In a further related embodimentis provided wherein the covered patch formed when the antigen bindingprotein is bound to human IL-23 comprises residue contact 121-123, 125and 283 of the human IL-23p40 subunit as described in SEQ ID NO:147.

Also provided is an isolated antigen binding protein that binds humanIL-23, wherein when said antigen binding protein is bound to human IL-23said antigen binding protein is 5 Å or less from a residue withinresidues 46-58, from a residue within residues 112-123, and from aresidue within residues 155-163 of the human IL-23p19 subunit asdescribed in SEQ ID NO:145, as determined by X-ray crystallography. Inone embodiment, when the antigen binding protein is bound to humanIL-23, the antigen binding protein is 5 Å or less from one, two, three,four, five, six, seven, eight, nine, ten, eleven, twelve or thirteenresidues within residues 46-58, from one, two, three, four, five, six,seven, eight, nine or ten, residues within residues 112-123, and fromone, two, three, four, five, six, seven, eight or nine residues withinresidues 155-163 of the human IL-23p19 subunit as described in SEQ IDNO:145. Within another embodiment when the antigen binding protein isbound to human IL-23 the antigen binding protein is 5 Å or less fromresidues 46-50, 113-116, 120, 156, 159, 160 and 163 of SEQ ID NO:145.Within another embodiment when the antigen binding protein is bound tohuman IL-23, the antigen binding protein is 5 Å or less from residues46-50, 112-120, 156, 159, 160 and 163 of SEQ ID NO:145. Within a relatedembodiment when the antigen binding protein is bound to human IL-23, theantigen binding protein is 5 Å or less from residues 46-50, 53, 112-120,156, 159, 160 and 163 of SEQ ID NO:145. Within another embodiment whenthe antigen binding protein is bound to human IL-23, the antigen bindingprotein is 5 Å or less from residues 46-50, 53-55, 58, 113-116, 120,121, 156, 159, 160, 162 and 163 of SEQ ID NO:145. Within a relatedembodiment when the antigen binding protein is bound to human IL-23, theantigen binding protein is 5 Å or less from residues 46-51, 53-55, 57,58, 112-116, 118-121, 123, 155, 156, 159, 160, 162 and 163 of SEQ IDNO:145. Within a further embodiment when the antigen binding protein isbound to human IL-23 the antigen binding protein is 5 Å or less from aresidue within residues 121-125, of the human IL-23p40 subunit asdescribed in SEQ ID NO:147, as determined by X-ray crystallography. Witha related embodiment when the antigen binding protein is bound to humanIL-23, said antigen binding protein is 5 Å or less from residues 122 and124 of SEQ ID NO:147. Within another embodiment when the antigen bindingprotein is bound to human IL-23, the antigen binding protein is 5 Å orless from residues 121-123 and 125 of SEQ ID NO:147.

Also provided is an isolated antigen binding protein as described above,wherein the antigen binding protein has at least one property selectedfrom the group consisting of: a) reducing human IL-23 activity; b)reducing production of a proinflammatory cytokine; c) binding to humanIL-23 with a KD of ≤5×10−8 M; d) having a Koff rate of ≤5×10−6 1/s; andd) having an IC50 of ≤400 pM.

A pharmaceutical composition comprising at least one antigen bindingprotein as described above and pharmaceutically acceptable excipient isprovided. In one embodiment is provided a pharmaceutical compositionfurther comprises a labeling group or an effector group. In yet anotherembodiment is provided a pharmaceutical composition wherein the labelinggroup is selected from the group consisting of isotopic labels, magneticlabels, redox active moieties, optical dyes, biotinylated groups andpredetermined polypeptide epitopes recognized by a secondary reporter.In yet another embodiment is provided a pharmaceutical compositionwherein the effector group is selected from the group consisting of aradioisotope, radionuclide, a toxin, a therapeutic group and achemotherapeutic group.

Also provided is a method for treating or preventing a conditionassociated with IL-23 in a patient, comprising administering to apatient in need thereof an effective amount of at least one isolatedantigen binding protein as described above. In one embodiment isprovided a method of wherein the condition is selected from the groupconsisting of an inflammatory disorder, a rheumatic disorder, anautoimmune disorder, an oncological disorder and a gastrointestinaldisorder. In yet another embodiment is provided a method wherein thecondition is selected from the group consisting of multiple sclerosis,rheumatoid arthritis, cancer, psoriasis, inflammatory bowel disease,Crohn's disease, ulcerative colitis, systemic lupus erythematosus,psoriatic arthritis, autoimmune myocarditis; type 1 diabetes andankylosing spondylitis. In still another embodiment is provided a methodwherein the isolated antigen-binding protein is administered alone or asa combination therapy.

Also provided is a method of reducing IL-23 activity in a patientcomprising administering an effective amount of at least one antigenbinding protein as described above. In one embodiment is provided amethod of reducing IL-23 activity, wherein said IL-23 activity isinducing production of a proinflammatory cytokine.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A: Results of STAT-luciferase reporter assay using recombinanthuman IL-23. All antibodies completely inhibited recombinant human IL-23

FIG. 1B: Results from STAT-luciferase reporter assay using native humanIL-23. Only half of those antibodies that completely inhibitedrecombinant human IL-23 were able to completely inhibit native humanIL-23

DETAILED DESCRIPTION

The present invention provides compositions, kits, and methods relatingto IL-23 antigen binding proteins, including molecules that antagonizeIL-23, such as anti-IL-23 antibodies, antibody fragments, and antibodyderivatives, e.g., antagonistic anti-IL-23 antibodies, antibodyfragments, or antibody derivatives. Also provided are polynucleotides,and derivatives and fragments thereof, comprising a sequence of nucleicacids that encodes all or a portion of a polypeptide that binds toIL-23, e.g., a polynucleotide encoding all or part of an anti-IL-23antibody, antibody fragment, or antibody derivative, plasmids andvectors comprising such nucleic acids, and cells or cell linescomprising such polynucleotides and/or vectors and plasmids. Theprovided methods include, for example, methods of making, identifying,or isolating IL-23 antigen binding proteins, such as anti-IL-23antibodies, methods of determining whether a molecule binds to IL-23,methods of determining whether a molecule antagonizes IL-23, methods ofmaking compositions, such as pharmaceutical compositions, comprising anIL-23 antigen binding protein, and methods for administering an IL-23antigen binding protein to a subject, for example, methods for treatinga condition mediated by IL-23, and for antagonizing a biologicalactivity of IL-23, in vivo or in vitro.

Unless otherwise defined herein, scientific and technical terms used inconnection with the present invention shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular. Generally,nomenclatures used in connection with, and techniques of, cell andtissue culture, molecular biology, immunology, microbiology, geneticsand protein and nucleic acid chemistry and hybridization describedherein are those well known and commonly used in the art. The methodsand techniques of the present invention are generally performedaccording to conventional methods well known in the art and as describedin various general and more specific references that are cited anddiscussed throughout the present specification unless otherwiseindicated. See, e.g., Sambrook et al., Molecular Cloning: A LaboratoryManual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (2001) and Ausubel et al., Current Protocols in MolecularBiology, Greene Publishing Associates (1992), and Harlow and LaneAntibodies: A Laboratory Manual Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y. (1990). Enzymatic reactions and purificationtechniques are performed according to manufacturer's specifications, ascommonly accomplished in the art or as described herein. The terminologyused in connection with, and the laboratory procedures and techniquesof, analytical chemistry, synthetic organic chemistry, and medicinal andpharmaceutical chemistry described herein are those well known andcommonly used in the art. Standard techniques can be used for chemicalsyntheses, chemical analyses, pharmaceutical preparation, formulation,and delivery, and treatment of patients.

All patents and other publications identified are expressly incorporatedherein by reference in their entirety for the purpose of describing anddisclosing, for example, the methodologies described in suchpublications that might be used in connection with information describedherein.

The polynucleotide and protein sequences of the p19 subunit of humanIL-23 (SEQ ID NOs: 144 and 145), the shared p40 subunit (SEQ ID NOs:146and 147), the human IL-23 receptor hererodimeric subunits IL-12Rβ1 (SEQID NOs: 150 and 151) and IL-23R (SEQ ID NOs: 148 and 149), are known inthe art, see for example, GenBank Accession Nos. AB030000; M65272,NM_005535, NM_144701, as are those from other mammalian species.Recombinant IL-23 and IL-23 receptor proteins including single chain andFc proteins as well as cells expressing the IL-23 receptor have beendescribed or are available from commercial sources. (see for example,Oppmann et al., Immunity, 2000, 13: 713-715; R&D Systems, Minneapolis.Minn.; United States Biological, Swampscott, Mass.; WIPO Publication No.WO 2007/076524). Native human IL-23 can be obtained from human cellssuch as dendritic cells using methods known in the art including thosedescribed herein.

IL-23 is a heterodimeric cytokine comprised of a unique p19 subunit thatis covalently bound to a shared p40 subunit. The p19 subunit comprisesfour α-helices, “A”, “B”, “C” and “D” in an up-up-down-down motif joinedby three intra-helix loops between the A and B helices, between the Band C helices and between the C and D helices, see Oppmann et al.,Immunity, 2000, 13: 713-715 and Beyer, et al., J Mol Biol, 2008. 382(4):942-55. The A and D helices of 4 helical bundle cytokines are believedto be involved with receptor binding. The p40 subunit comprises threebeta-sheet sandwich domains, D1, D2 and D3 (Lupardus and Garcia, J. Mol.Biol., 2008, 382:931-941.

The term “polynucleotide” includes both single-stranded anddouble-stranded nucleic acids and includes genomic DNA, RNA, mRNA, cDNA,or synthetic origin or some combination thereof which is not associatedwith sequences normally found in nature. Isolated polynucleotidescomprising specified sequences may include, in addition to the specifiedsequences, coding sequences for up to ten or even up to twenty otherproteins or portions thereof, or may include operably linked regulatorysequences that control expression of the coding region of the recitednucleic acid sequences, and/or may include vector sequences. Thenucleotides comprising the polynucleotide can be ribonucleotides ordeoxyribonucleotides or a modified form of either type of nucleotide.The modifications include base modifications such as bromouridine andinosine derivatives, ribose modifications such as 2′,3′-dideoxyribose,and internucleotide linkage modifications such as phosphorothioate,phosphorodithioate, phosphoroselenoate, phosphorodiselenoate,phosphoroanilothioate, phoshoraniladate and phosphoroamidate.

The term “oligonucleotide” means a polynucleotide comprising 100 orfewer nucleotides. In some embodiments, oligonucleotides are 10 to 60bases in length. In other embodiments, oligonucleotides are 12, 13, 14,15, 16, 17, 18, 19, or 20 to 40 nucleotides in length. Oligonucleotidesmay be single stranded or double stranded, e.g., for use in theconstruction of a mutant gene. Oligonucleotides may be sense orantisense oligonucleotides. An oligonucleotide can include a detectablelabel, such as a radiolabel, a fluorescent label, a hapten or anantigenic label, for detection assays. Oligonucleotides may be used, forexample, as PCR primers, cloning primers or hybridization probes.

The terms “polypeptide” or “protein” means a macromolecule having theamino acid sequence of a native protein, that is, a protein produced bya naturally-occurring and non-recombinant cell; or it is produced by agenetically-engineered or recombinant cell, and comprise moleculeshaving the amino acid sequence of the native protein, or moleculeshaving one or more deletions from, insertions to, and/or substitutionsof the amino acid residues of the native sequence. The term alsoincludes amino acid polymers in which one or more amino acids arechemical analogs of a corresponding naturally-occurring amino acid andpolymers. The terms “polypeptide” and “protein” encompass IL-23 antigenbinding proteins (such as antibodies) and sequences that have one ormore deletions from, additions to, and/or substitutions of the aminoacid residues of the antigen binding protein sequence. The term“polypeptide fragment” refers to a polypeptide that has anamino-terminal deletion, a carboxyl-terminal deletion, and/or aninternal deletion as compared with the full-length native protein. Suchfragments may also contain modified amino acids as compared with thenative protein. In certain embodiments, fragments are about five to 500amino acids long. For example, fragments may be at least 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 50, 70, 100, 110, 150, 200,250, 300, 350, 400, or 450 amino acids long. Useful polypeptidefragments include immunologically functional fragments of antibodies,including binding domains. In the case of an IL-23 antigen bindingprotein, such as an antibody, useful fragments include but are notlimited to one or more CDR regions, a variable domain of a heavy orlight chain, a portion of an antibody chain, a portion of a variableregion including less than three CDRs, and the like.

“Amino acid” includes its normal meaning in the art. The twentynaturally-occurring amino acids and their abbreviations followconventional usage. See, Immunology-A Synthesis, 2nd Edition, (E. S.Golub and D. R. Gren, eds.), Sinauer Associates: Sunderland, Mass.(1991). Stereoisomers (e.g., D-amino acids) of the twenty conventionalamino acids, unnatural amino acids such as [alpha]-,[alpha]-disubstituted amino acids, N-alkyl amino acids, and otherunconventional amino acids may also be suitable components forpolypeptides. Examples of unconventional amino acids include:4-hydroxyproline, [gamma]-carboxyglutamate,[epsilon]-N,N,N-trimethyllysine, [epsilon]-N-acetyllysine,O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine,5-hydroxylysine, [sigma]-N-methylarginine, and other similar amino acidsand imino acids (e.g., 4-hydroxyproline). In the polypeptide notationused herein, the left-hand direction is the amino terminal direction andthe right-hand direction is the carboxyl-terminal direction, inaccordance with standard usage and convention.

The term “isolated protein” refers to a protein, such as an antigenbinding protein (an example of which could be an antibody), that ispurified from proteins or polypeptides or other contaminants that wouldinterfere with its therapeutic, diagnostic, prophylactic, research orother use. As used herein, “substantially pure” means that the describedspecies of molecule is the predominant species present, that is, on amolar basis it is more abundant than any other individual species in thesame mixture. In certain embodiments, a substantially pure molecule is acomposition wherein the object species comprises at least 50% (on amolar basis) of all macromolecular species present. In otherembodiments, a substantially pure composition will comprise at least80%, 85%, 90%, 95%, or 99% of all macromolecular species present in thecomposition. In certain embodiments, an essentially homogeneoussubstance has been purified to such a degree that contaminating speciescannot be detected in the composition by conventional detection methodsand thus the composition consists of a single detectable macromolecularspecies.

A “variant” of a polypeptide (e.g., an antigen binding protein such asan antibody) comprises an amino acid sequence wherein one or more aminoacid residues are inserted into, deleted from and/or substituted intothe amino acid sequence relative to another polypeptide sequence.Variants include fusion proteins. A “derivative” of a polypeptide is apolypeptide that has been chemically modified in some manner distinctfrom insertion, deletion, or substitution variants, e.g., viaconjugation to another chemical moiety.

The terms “naturally occurring” or “native” as used throughout thespecification in connection with biological materials such aspolypeptides, nucleic acids, host cells, and the like, refers tomaterials which are found in nature, such as native human IL-23. Incertain aspects, recombinant antigen binding proteins that bind nativeIL-23 are provided. In this context, a “recombinant protein” is aprotein made using recombinant techniques, i.e., through the expressionof a recombinant nucleic acid as described herein. Methods andtechniques for the production of recombinant proteins are well known inthe art.

The term “antibody” refers to an intact immunoglobulin of any isotype,or a fragment thereof that can compete with the intact antibody forspecific binding to the target antigen, and includes, for instance,chimeric, humanized, fully human, and bispecific antibodies. An antibodyas such is a species of an antigen binding protein. Unless otherwiseindicated, the term “antibody” includes, in addition to antibodiescomprising two full-length heavy chains and two full-length lightchains, derivatives, variants, fragments, and muteins thereof, examplesof which are described below. An intact antibody generally will compriseat least two full-length heavy chains and two full-length light chains,but in some instances may include fewer chains such as antibodiesnaturally occurring in camelids which may comprise only heavy chains.Antibodies may be derived solely from a single source, or may be“chimeric,” that is, different portions of the antibody may be derivedfrom two different antibodies as described further below. The antigenbinding proteins, antibodies, or binding fragments may be produced inhybridomas, by recombinant DNA techniques, or by enzymatic or chemicalcleavage of intact antibodies.

The term “functional fragment” (or simply “fragment”) of an antibody orimmunoglobulin chain (heavy or light chain), as used herein, is anantigen binding protein comprising a portion (regardless of how thatportion is obtained or synthesized) of an antibody that lacks at leastsome of the amino acids present in a full-length chain but which iscapable of specifically binding to an antigen. Such fragments arebiologically active in that they bind specifically to the target antigenand can compete with other antigen binding proteins, including intactantibodies, for specific binding to a given epitope. In one aspect, sucha fragment will retain at least one CDR present in the full-length lightor heavy chain, and in some embodiments will comprise a single heavychain and/or light chain or portion thereof. These biologically activefragments may be produced by recombinant DNA techniques, or may beproduced by enzymatic or chemical cleavage of antigen binding proteins,including intact antibodies. Fragments include, but are not limited to,immunologically functional fragments such as Fab, Fab′, F(ab′)2, Fv,domain antibodies and single-chain antibodies, and may be derived fromany mammalian source, including but not limited to human, mouse, rat,camelid or rabbit. It is contemplated further that a functional portionof the antigen binding proteins disclosed herein, for example, one ormore CDRs, could be covalently bound to a second protein or to a smallmolecule to create a therapeutic agent directed to a particular targetin the body, possessing bifunctional therapeutic properties, or having aprolonged serum half-life.

The term “compete” when used in the context of antigen binding proteins(e.g., neutralizing antigen binding proteins or neutralizing antibodies)means competition between antigen binding proteins as determined by anassay in which the antigen binding protein (e.g., antibody orimmunologically functional fragment thereof) under test prevents orinhibits specific binding of a reference antigen binding protein (e.g.,a ligand, or a reference antibody) to a common antigen (e.g., an IL-23protein or a fragment thereof). Numerous types of competitive bindingassays can be used, for example: solid phase direct or indirectradioimmunoassay (RIA), solid phase direct or indirect enzymeimmunoassay (EIA), sandwich competition assay (see, e.g., Stahli et al.,1983, Methods in Enzymology 92:242-253); solid phase directbiotin-avidin EIA (see, e.g., Kirkland et al., 1986, J. Immunol.137:3614-3619) solid phase direct labeled assay, solid phase directlabeled sandwich assay (see, e.g., Harlow and Lane, 1988, Antibodies, ALaboratory Manual, Cold Spring Harbor Press); solid phase direct labelRIA using 1-125 label (see, e.g., Morel et al., 1988, Molec. Immunol.25:7-15); solid phase direct biotin-avidin EIA (see, e.g., Cheung, etal., 1990, Virology 176:546-552); and direct labeled RIA (Moldenhauer etal., 1990, Scand. J. Immunol. 32:77-82). Typically, such an assayinvolves the use of purified antigen bound to a solid surface or cellsbearing either of these, an unlabelled test antigen binding protein anda labeled reference antigen binding protein.

Competitive inhibition is measured by determining the amount of labelbound to the solid surface or cells in the presence of the test antigenbinding protein. Usually the test antigen binding protein is present inexcess. Antigen binding proteins identified by competition assay(competing antigen binding proteins) include antigen binding proteinsbinding to the same epitope as the reference antigen binding proteinsand antigen binding proteins binding to an adjacent epitope sufficientlyproximal to the epitope bound by the reference antigen binding proteinfor steric hindrance to occur. Usually, when a competing antigen bindingprotein is present in excess, it will inhibit specific binding of areference antigen binding protein to a common antigen by at least 40%,45%, 50%, 55%, 60%, 65%, 70% or 75%. In some instance, binding isinhibited by at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%98%, 99% or more.

The term “epitope” or “antigenic determinant” refers to a site on anantigen to which an antigen binding protein binds. Epitopes can beformed both from contiguous amino acids or noncontiguous amino acidsjuxtaposed by tertiary folding of a protein. Epitopes formed fromcontiguous amino acids are typically retained on exposure to denaturingsolvents, whereas epitopes formed by tertiary folding are typically loston treatment with denaturing solvents. Epitope determinants may includechemically active surface groupings of molecules such as amino acids,sugar side chains, phosphoryl or sulfonyl groups, and may have specificthree dimensional structural characteristics, and/or specific chargecharacteristics. An epitope typically includes at least 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35 amino acidsin a unique spatial conformation. Epitopes can be determined usingmethods known in the art.

IL-23 Antigen Binding Proteins

An “antigen binding protein” as used herein means a protein thatspecifically binds a specified target antigen; the antigen as providedherein is IL-23, particularly human IL-23, including native human IL-23.Antigen binding proteins as provided herein interact with at least aportion of the unique p19 subunit of IL-23, detectably binding IL-23;but do not bind with any significance to IL-12 (e.g., the p40 and/or thep35 subunits of IL-12), thus “sparing IL-12”. As a consequence, theantigen binding proteins provided herein are capable of impacting IL-23activity without the potential risks that inhibition of IL-12 or theshared p40 subunit might incur. The antigen binding proteins may impactthe ability of IL-23 to interact with its receptor, for example byimpacting binding to the receptor, such as by interfering with receptorassociation. In particular, such antigen binding proteins totally orpartially reduce, inhibit, interfere with or modulate one or morebiological activities of IL-23. Such inhibition or neutralizationdisrupts a biological response in the presence of the antigen bindingprotein compared to the response in the absence of the antigen bindingprotein and can be determined using assays known in the art anddescribed herein. Antigen binding proteins provided herein inhibitIL-23-induced proinflammatory cytokine production, for exampleIL-23-induced IL-22 production in whole blood cells and IL-23-inducedIFNγ expression in NK and whole blood cells. Reduction of biologicalactivity can be about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more.

An antigen binding protein may comprise a portion that binds to anantigen and, optionally, a scaffold or framework portion that allows theantigen binding portion to adopt a conformation that promotes binding ofthe antigen binding protein to the antigen. Examples of antigen bindingproteins include antibodies, antibody fragments (e.g., an antigenbinding portion of an antibody), antibody derivatives, and antibodyanalogs. The antigen binding protein can comprise an alternative proteinscaffold or artificial scaffold with grafted CDRs or CDR derivatives.Such scaffolds include, but are not limited to, antibody-derivedscaffolds comprising mutations introduced to, for example, stabilize thethree-dimensional structure of the antigen binding protein as well aswholly synthetic scaffolds comprising, for example, a biocompatiblepolymer. See, for example, Korndorfer et al., Proteins: Structure,Function, and Bioinformatics, (2003) Volume 53, Issue 1:121-129; Roqueet al., Biotechnol. Prog., 2004, 20:639-654. In addition, peptideantibody mimetics (“PAMs”) can be used, as well as scaffolds based onantibody mimetics utilizing fibronection components as a scaffold.

Certain antigen binding proteins described herein are antibodies or arederived from antibodies. Such antigen binding proteins include, but arenot limited to, monoclonal antibodies, bispecific antibodies,minibodies, domain antibodies, synthetic antibodies, antibody mimetics,chimeric antibodies, humanized antibodies, human antibodies, antibodyfusions, antibody conjugates, single chain antibodies, and fragmentsthereof, respectively. In some instances, the antigen binding protein isan immunological fragment of an antibody (e.g., a Fab, a Fab′, aF(ab′)2, or a scFv). The various structures are further described anddefined herein.

Certain antigen binding proteins that are provided may comprise one ormore CDRs as described herein (e.g., 1, 2, 3, 4, 5, 6 or more CDRs). Insome instances, the antigen binding protein comprises (a) a polypeptidestructure and (b) one or more CDRs that are inserted into and/or joinedto the polypeptide structure. The polypeptide structure can take avariety of different forms. For example, it can be, or comprise, theframework of a naturally occurring antibody, or fragment or variantthereof, or may be completely synthetic in nature. Examples of variouspolypeptide structures are further described below.

An antigen binding protein of the invention is said to “specificallybind” its target antigen when the dissociation equilibrium constant (KD)is ≤10−8 M. The antigen binding protein specifically binds antigen with“high affinity” when the KD is ≤5×10−9 M, and with “very high affinity”when the the KD is ≤5×10−10 M. In one embodiment the antigen bindingprotein will bind to human IL-23 with a KD of ≤5×10−12 M, and in yetanother embodiment it will bind with a KD≤5×10−13 M. In anotherembodiment of the invention, the antigen binding protein has a KD of≤5×10−12 M and an Koff of about ≤5×10−6 1/s. In another embodiment, theKoff is ≤5×10−7 1/s.

Another aspect provides an antigen binding protein having a half-life ofat least one day in vitro or in vivo (e.g., when administered to a humansubject). In one embodiment, the antigen binding protein has a half-lifeof at least three days. In another embodiment, the antibody or portionthereof has a half-life of four days or longer. In another embodiment,the antibody or portion thereof has a half-life of eight days or longer.In another embodiment, the antibody or antigen binding portion thereofis derivatized or modified such that it has a longer half-life ascompared to the underivatized or unmodified antibody. In anotherembodiment, the antigen binding protein contains point mutations toincrease serum half life, such as described in WIPO Publication No. WO00/09560.

In embodiments where the antigen binding protein is used for therapeuticapplications, an antigen binding protein can reduce, inhibit, interferewith or modulate one or more biological activities of IL-23, suchinducing production of proinflammatory cytokines. IL-23 has manydistinct biological effects, which can be measured in many differentassays in different cell types; examples of such assays and known andare provided herein.

Some of the antigen binding proteins that are provided have thestructure typically associated with naturally occurring antibodies. Thestructural units of these antibodies typically comprise one or moretetramers, each composed of two identical couplets of polypeptidechains, though some species of mammals also produce antibodies havingonly a single heavy chain. In a typical antibody, each pair or coupletincludes one full-length “light” chain (in certain embodiments, about 25kDa) and one full-length “heavy” chain (in certain embodiments, about50-70 kDa). Each individual immunoglobulin chain is composed of several“immunoglobulin domains”, each consisting of roughly 90 to 110 aminoacids and expressing a characteristic folding pattern. These domains arethe basic units of which antibody polypeptides are composed. Theamino-terminal portion of each chain typically includes a variableregion that is responsible for antigen recognition. The carboxy-terminalportion is more conserved evolutionarily than the other end of the chainand is referred to as the “constant region” or “C region”. Human lightchains generally are classified as kappa and lambda light chains, andeach of these contains one variable region and one constant domain(CL1).z Heavy chains are typically classified as mu, delta, gamma,alpha, or epsilon chains, and these define the antibody's isotype asIgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subtypes,including, but not limited to, IgG1, IgG2, IgG3, and IgG4. IgM subtypesinclude IgM, and IgM2. IgA subtypes include IgA1 and IgA2. In humans,the IgA and IgD isotypes contain four heavy chains and four lightchains; the IgG and IgE isotypes contain two heavy chains and two lightchains; and the IgM isotype contains five heavy chains and five lightchains. The heavy chain constant region (CH) typically comprises one ormore domains that may be responsible for effector function. The numberof heavy chain constant region domains will depend on the isotype. IgGheavy chains, for example, each contains three CH region domains knownas CH1, CH2 and CH3. The antibodies that are provided can have any ofthese isotypes and subtypes, for example, the IL-23 antigen bindingprotein is of the IgG1, IgG2, or IgG4 subtype. If an IgG4 is desired, itmay also be desired to introduce a point mutation (CPSCP→CPPCP) in thehinge region as described in Bloom et al., 1997, Protein Science 6:407)to alleviate a tendency to form intra-H chain disulfide bonds that canlead to heterogeneity in the IgG4 antibodies. Antibodies provided hereinthat are of one type can be changed to a different type using subclassswitching methods. See, e.g., Lantto et al., 2002, Methods Mol. Biol.178:303-316.

In full-length light and heavy chains, the variable and constant regionsare joined by a “J” region of about twelve or more amino acids, with theheavy chain also including a “D” region of about ten more amino acids.See, e.g., Fundamental Immunology, 2nd ed., Ch. 7 (Paul, W., ed.) 1989,New York: Raven Press. The variable regions of each light/heavy chainpair typically form the antigen binding site.

Variable Regions

Various heavy chain and light chain variable regions (or domains)provided herein are depicted in TABLES 1 and 2. Each of these variableregions may be attached, for example, to heavy and light chain constantregions described above. Further, each of the so generated heavy andlight chain sequences may be combined to form a complete antigen bindingprotein structure.

Provided are antigen binding proteins that contain at least one heavychain variable region (VH) selected from the group consisting of VH1,VH2, VH3, VH4, VH5, VH6, VH7, VH8, VH9, VH10, VH11, VH12, VH13, VH14,VH15 and VH16 and/or at least one light chain variable region (VL)selected from the group consisting of VL1, VL2, VL3, VL4, VL5, VL6, VL7,VL8, VL9, VL10, VL11, VL12, VL13, VL14, VL15, and VL16 as shown inTABLES 1 and 2 below.

Each of the heavy chain variable regions listed in TABLE 2 may becombined with any of the light chain variable regions shown in TABLE 1to form an antigen binding protein. In some instances, the antigenbinding protein includes at least one heavy chain variable region and/orone light chain variable region from those listed in TABLES 1 and 2. Insome instances, the antigen binding protein includes at least twodifferent heavy chain variable regions and/or light chain variableregions from those listed in TABLES 1 and 2. The various combinations ofheavy chain variable regions may be combined with any of the variouscombinations of light chain variable regions.

In other instances, the antigen binding protein contains two identicallight chain variable regions and/or two identical heavy chain variableregions. As an example, the antigen binding protein may be an antibodyor immunologically functional fragment that comprises two light chainvariable regions and two heavy chain variable regions in combinations ofpairs of light chain variable regions and pairs of heavy chain variableregions as listed in TABLES 1 and 2. Examples of such antigen bindingproteins comprising two identical heavy chain and light chain variableregions include: Antibody A VH14/VL14; Antibody B VH9/VL9; Antibody CVH10/VL10; Antibody D VH15/VL15; Antibody E VH1/VL1, Antibody FVH11/VL11; Antibody G VH12/VL12; Antibody H VH13/VL13; Antibody IVH8/VL8; Antibody J VH3/VL3; Antibody K VH7/VL7; Antibody L VH4/VL4;Antibody M VH5/VL5 and Antibody N VH6/VL6.

Some antigen binding proteins that are provided comprise a heavy chainvariable region and/or a light chain variable region comprising asequence of amino acids that differs from the sequence of a heavy chainvariable region and/or a light chain variable region selected fromTABLES 1 and 2 at only 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or15 amino acid residues, wherein each such sequence difference isindependently either a deletion, insertion or substitution of one aminoacid. The light and heavy chain variable regions, in some antigenbinding proteins, comprise sequences of amino acids that have at least70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%sequence identity to the amino acid sequences provided in TABLES 1 and2. Still other antigen binding proteins, e.g., antibodies orimmunologically functional fragments, also include variant heavy chainregion forms and/or variant light chain region forms as describedherein.

The term “identity” refers to a relationship between the sequences oftwo or more polypeptide molecules or two or more polynucleotides, asdetermined by aligning and comparing the sequences. “Percent identity”means the percent of identical residues between the amino acids ornucleotides in the compared molecules and is calculated based on thesize of the smallest of the molecules being compared.

TABLE 1 Exemplary Variant Light Chain Region SequencesFR1                       CDRL1          FR2         CDRL2          FR3                        CDRL3       FR4V_(L)1 QSVLTQPPSVSGAPGQRVTISC

WYQQVPGTAPKLLIY

GVPDRFSGSKSGTSASLAITGLQAEDEADYYC

GGGTRLTVL SEQ ID NO: 1 V_(L)2 QSVLTQPPSVSGAPGQRVTISC

WYQQLPGTAPKLLIY

GVPDRFSGSKSGTSASLAITGLQAEDEADYYC

GGGTKLTVL SEQ ID NO: 3 V_(L)3 QAVLTQPSSLSASPGASASLTC

WYQQKPGSPPQYLLR

GVPSRFSGSKDASANAGILLISGLQSEDEADYYC

FGGGTKLTVL SEQ ID NO: 4 V_(L)4 QAVLTQPSSLSASPGASASLTC

WYQQKPGSPPQYLLR

GVPSRFSGSKDASANAGILLISGLQSEDEADYYC

FGGGTKLTVL SEQ ID NO: 4 V_(L)5 QPVLTQPPSASASLGASVTLTC

WYQQRPGKGPRFVMR

GIPDRFSVLGSGLNRYLTIKNIQEEDESDYHC

FGTGTKVTVL SEQ ID NO 7 V_(L)6 QPVLTQPPSASASLGASVTLTC

WYQQRPGKGPRFVMR

GIPDRFSVLGSGLNRYLTIKNIQEEDESDYHC

FGTGTKVTVL SEQ ID NO: 9 V_(L)7 QPELTQPPSASASLGASVTLTC

WYQLRPGKGPRFVMR

GIPDRFSVLGSGLNRSLTIKNIQEEDESDYHC

FGTGTKVTVL SEQ ID NO: 11 V_(L)8 DIQLTPSPSSVSASVGDRVTITC

WYQQKPGKAPKLLIY

GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC

FGGGTKVEIK SEQ ID NO: 13 V_(L)9 DIQMTQSPSSVSASVGDRVTITC

WYQQKPGKAPSLLIY

GVPSRFSGSVSGTDFTLTISSLQPEDFATYYC

FGPGTKVDFK SEQ ID NO: 15 V_(L)10 DIQMTQSPSSVSASVGDRVTITC

WYQQKPGKAPKLLIY

GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC

FGPGTKVDIK SEQ ID NO: 17 V_(L)11 DSQMTQSPSSVSASVGDRVTITC

WYQQKPGQAPNLLIY

GVPSRFSGSGSGTEFTLTISSLQPEDFATYYC

FGPGTKVDIK SEQ ID NO: 19 V_(L)12 DIQMTQSPSSVSASVGDRVTITC

WYQQKPGKAPKLLIY

GVPSRFSGSGSGTDFTLTISSLQPDDFATYYC

GGGTKVEIK SEQ ID NO: 21 V_(L)13 DIQMTQSPSSVSASVGDRVTITC

WYQQKPGKAPKLLIY

GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC

FGPGTKVDIK SEQ ID NO: 23 V_(L)14 DIQLTQSPSSVSASVGDRVTITC

WYQQKPGKAPNLLIY

GVPSRFSGSGSGTDFTLTISSLQPADFATYFC

FGPGTKVDVK SEQ ID NO: 25 V_(L)15 DIQMTQSPSSVSASVGDRVTITC

WYQQKPGKAPKLLIY

GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC

FGPGTKVDIK SEQ ID NO: 27 V_(L)16 DIQMTQSPSSLSASVGDRVTITC

WYQQKPGKAPKRLIY

GVPSRFSGSGSGTEFTLTISSLQPEDFATYYC

FGQGTKVEIE SEQ ID NO: 29

TABLE 2 Exemplary Variant Heavy Chain Region Sequences                   FR1               CDRH1       FR2           CDRH2                    FR3                    CDRH3            FR4V_(H)1 QVQLVESGGGVVQPGRSLRLSCAASGFTFS

WVRQAPGKGLEWVA

RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR

WGQGTMVTVSS SEQ ID NO: 31 V_(H)2 QVQLVESGGGVVQPGRSLRLSCAASGFTFS

WVRQAPGKGLEWVA

RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR

WGQGTMVTVSS SEQ ID NO: 33 V_(H)3 QVQLVESGGGVVQPGRSLRLSCAASGFTFS

WVRQAPGKGLEWVA

RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR

WGQGTLVTVSS SEQ ID NO: 34 V_(H)4 QVQLVESGGGVVQPGRSLRLSCAASGFTFS

WVRQAPGKGLEWLS

RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR

WGQGTLVTVSS SEQ ID NO: 36 V_(H)5 EVQLVESGGGLVQPGGSLRLSCAASGFTFS

WVRQAPGKGLEWVS

RFTISRDNAKNSLYLQMNSLRDEDTAVYYCAR

WGQGTIVTVSS SEQ ID NO: 38 V_(H)6 EVQLVESGGGLVQPGGSLRLSCAASGFTFS

WRQAPGKGLEWVS

RFTISRDNAKNSLYLQMNSLRDEDTAVYYCAR

WGQGTTVTVSS SEQ ID NO: 40 V_(H)7 EVQLVESGGGLVQPGGSLRLSCVVSGFTFS

WVRQAPGKGLEWVS

RFTISRDNAKNSLYLQMNSLRDEDTAVYYCAR

WGQGTTVTVSS SEQ ID NO: 42 V_(H)8 QVQLQESGPGLVKPSETLSLTCTVSGGSIS

WIRQPAGKGLEWIG

RVTMSLDTSKNQFSLRLTSVTAADTAVYYCAR

WGQGTTNITVSS SEQ ID NO: 44 V_(H)9 QVQLQESGPGLVKPSQTLSLTCTVSGGSIS

WIRQHPGKGLEWIG

RVTISVDTSKNQFSLKLSSVTAADTAVYYCAK

WGQGTTVIVSS SEQ ID NO: 46 V_(H)10  QVQLQESGPGLVKPSQTLSLTCTVSGGSIN

WIRQHPGKGLEWIG

RVTISVDTSQNQFSLKLSSVTAADTAVYYCAR

WGQGTTVTVSS SEQ ID NO: 48 V_(H)11  QVQLQESGPGLVKPSQTLSLTCTVSGGSI

WIRQHPGKGLEWIG

RVTISVDTSKNQFSLKLSSVTAADTAVYYCAR

WGQGTTVIVSS SEQ ID NO: 50 V_(H)12  QVQLQESGPRLVKPSETLSLTCTVSGDSIS

WIRQPPGKGLEWLG

RVTISIDTSKNQFSLKLSSVTAADTAVYYCTR

WGQGTLVTVSS SEQ ID NO: 52 V_(H)13  QVQLQESGPGLVKPSQTLSLICTVSGGSIS

WIRQHPGKGLEWIG

RITISVDTSKNQFSLSLSSVTAADTAVYYCAR

WGQGTTVIVSS SEQ ID NO: 54 V_(H)14  QVQLQESGPGLVKPSQTLSLTCTVSGGSIS

WIRQHPGKGLEWIG

RVIMSVDTSKNQFSLKLSSVTAADTAVYYCAK

WGQGTTVTVSS SEQ ID NO: 56 V_(H)15  QVQLQESGPGLVKPSQTLSLTCTVSGGSIN

WIRQHPGKGLEWIG

RVTISVDTSKNQFSLKLSSVTAADTAVYYCAR

WGQGTTVTVSS SEQ ID NO: 58 V_(H)16  QVQLVESGGGVVQPGRSLRLSCAASGFTFS

WVRQAPGKGLEWVA

RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR

WGQGTTVTVSS SEQ ID NO: 60For these calculations, gaps in alignments (if any) must be addressed bya particular mathematical model or computer program (i.e., an“algorithm”). Methods that can be used to calculate the identity of thealigned nucleic acids or polypeptides include those described inComputational Molecular Biology, (Lesk, A. M., ed.), 1988, New York:Oxford University Press; Biocomputing Informatics and Genome Projects,(Smith, D. W., ed.), 1993, New York: Academic Press; Computer Analysisof Sequence Data, Part I, (Griffin, A. M., and Griffin, H. G., eds.),1994, New Jersey: Humana Press; von Heinje, G., 1987, Sequence Analysisin Molecular Biology, New York: Academic Press; Sequence AnalysisPrimer, (Gribskov, M. and Devereux, J., eds.), 1991, New York: M.Stockton Press; and Carillo et al., 1988, SIAM J. Applied Math. 48:1073.

In calculating percent identity, the sequences being compared arealigned in a way that gives the largest match between the sequences. Thecomputer program used to determine percent identity is the GCG programpackage, which includes GAP (Devereux et al., 1984, Nucl. Acid Res.12:387; Genetics Computer Group, University of Wisconsin, Madison,Wis.). The computer algorithm GAP is used to align the two polypeptidesor polynucleotides for which the percent sequence identity is to bedetermined. The sequences are aligned for optimal matching of theirrespective amino acid or nucleotide (the “matched span”, as determinedby the algorithm). A gap opening penalty (which is calculated as 3× theaverage diagonal, wherein the “average diagonal” is the average of thediagonal of the comparison matrix being used; the “diagonal” is thescore or number assigned to each perfect amino acid match by theparticular comparison matrix) and a gap extension penalty (which isusually 1/10 times the gap opening penalty), as well as a comparisonmatrix such as PAM 250 or BLOSUM 62 are used in conjunction with thealgorithm. In certain embodiments, a standard comparison matrix (see,Dayhoff et al., 1978, Atlas of Protein Sequence and Structure 5:345-352for the PAM 250 comparison matrix; Henikoff et al., 1992, Proc. Natl.Acad. Sci. U.S.A. 89:10915-10919 for the BLOSUM 62 comparison matrix) isalso used by the algorithm.

Recommended parameters for determining percent identity for polypeptidesor nucleotide sequences using the GAP program are the following:Algorithm: Needleman et al., 1970, J. Mol. Biol. 48:443-453; Comparisonmatrix: BLOSUM 62 from Henikoff et al., 1992, supra; Gap Penalty: 12(but with no penalty for end gaps), Gap Length Penalty: 4, Threshold ofSimilarity: 0. Certain alignment schemes for aligning two amino acidsequences may result in matching of only a short region of the twosequences and this small aligned region may have very high sequenceidentity even though there is no significant relationship between thetwo full-length sequences. Accordingly, the selected alignment method(GAP program) can be adjusted if so desired to result in an alignmentthat spans at least 50 contiguous amino acids of the target polypeptide.

The heavy and light chain variable regions disclosed herein includeconsensus sequences derived from groups of related antigen bindingproteins. The amino acid sequences of the heavy and light chain variableregions were analyzed for similarities. Four groups emerged, one grouphaving kappa light chain variable regions, (V_(H)9/V_(L)9,V_(H)10/V_(L)10, V_(H)11/V_(L)11, V_(H)13/V_(L)13, V_(H)14/V_(L)14 andV_(H)15/V_(L)15) and three groups having lambda light chain variableregions: lambda group 1 (V_(H)5/V_(L)5, V_(H)6/V_(L)6 andV_(H)7/V_(L)7), lambda group 2 (V_(H)3/V_(L)3 and V_(H)4/V_(L)4), andlambda group 3 (V_(H)1/V_(L)1 and V_(H)2/V_(L)2). Light chain germlinesrepresented include VK1/A30 and VK1/L19. Light chain lambda germlinesrepresented include VL1/1e, VL3/3p, VL5/5c and VL9/9a. Heavy chaingermlines represented include VH3/3-30, VH3/3-30.3, VH3/3-33, VH3/3-48,VH4/4-31 and VH4/4-59. As used herein, a “consensus sequence” refers toamino acid sequences having conserved amino acids common among a numberof sequences and variable amino acids that vary within given amino acidsequences. Consensus sequences may be determined using standardphylogenic analyses of the light and heavy chain variable regionscorresponding to the IL-23 antigen binding proteins disclosed herein.

The light chain variable region consensus sequence for the kappa groupisDX₁QX₂TQSPSSVSASVGDRVTITCRASQGX₃X₄SX₅WX₆AWYQQKPGX₇APX₈LLIYAASSLQSGVPSRFS GSX₉SGTX₁₀FTLTISSLQPX₁₁DFATYX₁₂CQQANSFPFTFGPGTKVDX₁₃K (SEQ ID NO:30)where X₁ is selected from I or S; X₂ is selected from M or L; X₃ isselected from G or V and X₄ is selected from S, F or I; X₅ is selectedfrom S or G; X₆ is selected from F or L; X₇ is selected from K or Q; X₈is selected from K, N or S; X₉ is selected from G or V; X₁₀ is selectedfrom D or E, X₁₁ is selected from E or A; X₁₂ is selected from Y or F;and X₁₃ is selected from I, V or F.

The light chain variable region consensus sequence for lambda group 1 isQPX₁LTQPPSASASLGASVTLTCTLX₂SGYSDYKVDWYQX₃RPGKGPRFVMRVGTGGX₄VGSKGX₅GIPDRFSVLGSGLNRX₆LTIKNIQEEDESDYHCGADHGSGX₇NFVYVFGTGTKVTVL (SEQ ID NO:61)where X₁ is selected from V or E; X₂ is selected from N or S; X₃ isselected from Q or L and X₄ is selected from I or T; X₅ is selected fromD or E; X₆ is selected from Y or S; and X₇ is selected from S or N.

The light chain variable region consensus sequence for lambda group 3 isQSVLTQPPSVSGAPGQRVTISCTGSSSNX₁GAGYDVHWYQQX₂PGTAPKLLIYGSX₃NRPSGVPDRF SGSKSGTSASLAITGLQAEDEADYYCQSYDSSLSGWVFGGGTX₄RLTVL (SEQ ID NO:139) where X₁is selected from T or I; X₂ is selected from V or L; X₃ is selected fromG or N and X₄ is selected from R or K.

The heavy chain variable region consensus sequence for the kappa groupis QVQLQESGPGLVKPSQTLSLTCTVSGGSIX₁SGGYYWX₂WIRQHPGKGLEWIGX₃IX₄YSGX₅X₆YYNPSLK SRX₇TX₈SVDTSX₉NQFSLX₁₀LSSVTAADTAVYYCAX₁₁X₁₂RGX₁₃YYGMDVWGQGTTVTVSS(SEQ ID NO:140) where X₁ is selected from N or S; X₂ is selected from Sor T; X₃ is selected from Y or H and X₄ is selected from Y or H; X₅ isselected from S or N; X₆ is selected from S or T; X₇ is selected from Vor I; X₈ is selected from I or M; X₉ is selected from K or Q; X₁₀ isselected from K or S, X₁₁ is selected from R or K; X₁₂ is selected fromD or N; and X₁₃ is selected from H, F or Y.

The heavy chain variable region consensus sequence for lambda group 1 isEVQLVESGGGLVQPGGSLRLSCX₁X₂SGFTFSX₃X₄SMNWVRQAPGKGLEWVSYISSX₅SSTX₆YX₇AD SVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCARRIAAAGX₈X₉X₁₀YYYAX₁₁DVWGQGTTVTVSS (SEQID NO:141) where X₁ is selected from A or V; X₂ is selected from A or V;X₃ is selected from T or S and X₄ is selected from Y or F; X₅ isselected from S or R; X₆ is selected from R or I; X₇ is selected from H,Y or I; X₈ is selected from P or G; X₉ is selected from W or F; X₁₀ isselected from G or H and X₁₁ is selected from M or L.

The heavy chain variable region consensus sequence for lambda group 2 isQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYX₁MHWVRQAPGKGLEWX₂X₃VISX₄DGSX₅KYYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARERTTLSGSYFDYWGQGTLVTVSS (SEQ IDNO:142) where X₁ is selected from G or A; X₂ is selected from V or L; X₃is selected from A or S and X₄ is selected from F or H and X₅ isselected from L or I.

The heavy chain variable region consensus sequence for lambda group 3 isQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDGSNX₁YYADSV KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRGYX₂SSWYPDAFDIWGQGTMVTVSS (SEQ ID NO:143) where X₁ is selected from E or K and X₂ is selected from T or S.

Complementarity Determining Regions

Complementarity determining regions or “CDRs” are embedded within aframework in the heavy and light chain variable regions where theyconstitute the regions responsible for antigen binding and recognition.Variable domains of immunoglobulin chains of the same species, forexample, generally exhibit a similar overall structure; comprisingrelatively conserved framework regions (FR) joined by hypervariable CDRregions. An antigen binding protein can have 1, 2, 3, 4, 5, 6 or moreCDRs. The variable regions discussed above, for example, typicallycomprise three CDRs. The CDRs from heavy chain variable regions andlight chain variable regions are typically aligned by the frameworkregions to form a structure that binds specifically on a target antigen(e.g., IL-23). From N-terminal to C-terminal, naturally-occurring lightand heavy chain variable regions both typically conform to the followingorder of these elements: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. TheCDR and FR regions of exemplary light chain variable domains and heavychain variable domains are highlighted in TABLES 1 and 2. It isrecognized that the boundaries of the CDR and FR regions can vary fromthose highlighted. Numbering systems have been devised for assigningnumbers to amino acids that occupy positions in each of these domains.Complementarity determining regions and framework regions of a givenantigen binding protein may be identified using these systems. Numberingsystems are defined in Kabat et al., Sequences of Proteins ofImmunological Interest, 5^(th) Ed., US Dept. of Health and HumanServices, PHS, NIH, NIH Publication No. 91-3242, 1991, or Chothia &Lesk, 1987, J. Mol. Biol. 196:901-917; Chothia et al., 1989, Nature342:878-883. Other numbering systems for the amino acids inimmunoglobulin chains include IMGT® (the international ImMunoGeneTicsinformation system; Lefranc et al, Dev. Comp. Immunol. 2005,29:185-203); and AHo (Honegger and Pluckthun, J. Mol. Biol. 2001,309(3):657-670). The CDRs provided herein may not only be used to definethe antigen binding domain of a traditional antibody structure, but maybe embedded in a variety of other polypeptide structures, as describedherein.

The antigen binding proteins disclosed herein are polypeptides intowhich one or more CDRs may be grafted, inserted, embedded and/or joined.An antigen binding protein can have, for example, one heavy chain CDR1(“CDRH1”), and/or one heavy chain CDR2 (“CDRH2”), and/or one heavy chainCDR3 (“CDRH3”), and/or one light chain CDR1 (“CDRL1”), and/or one lightchain CDR2 (“CDRL2”), and/or one light chain CDR3 (“CDRL3”). Someantigen binding proteins include both a CDRH3 and a CDRL3. Specificembodiments generally utilize combinations of CDRs that arenon-repetitive, e.g., antigen binding proteins are generally not madewith two CDRH2 regions in one variable heavy chain region, etc. Antigenbinding proteins may comprise one or more amino acid sequences that areidentical to or that differ from to the amino acid sequences of one ormore of the CDRs presented in TABLE 3 at only 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14 or 15 amino acid residues, wherein each such sequencedifference is independently either a deletion, insertion or substitutionof one amino acid. The CDRs in some antigen binding proteins comprisesequences of amino acids that have at least 80%, 85%, 90%, 91%, 92, 93%,94%, 95%, 96%, 97%, 98%, or 99% sequence identity to CDRs sequencelisted in TABLE 3. In some antigen binding proteins, the CDRs areembedded into a “framework” region, which orients the CDR(s) such thatthe proper antigen binding properties of the CDR(s) is achieved.

TABLE 3 Exemplary CDRH and CDRL Sequences Exemplary CDRL Sequences CDRL1CDRL2 CDRL3 TGSSSNTGAGYDVH GSGN RPS QSYDSSLSGWV SEQ ID NO: 62SEQ ID NO: 63 SEQ ID NO: 64 TGSSSNIGAGYDVH GSNNRPS MIWHSSASVSEQ ID NO: 65 SEQ ID NO: 66 SEQ ID NO: 67 TLRSGINVGTYRIY YKSDSDKQQGSGADHGSGSNFVYV SEQ ID NO: 68 SEQ ID NO: 69 SEQ ID NO: 70 TLNSGYSDYKVVGTGGIVGSKGD GADHGSGNNFVYV SEQ ID NO: 71 SEQ ID NO: 72 SEQ ID NO: 73TLSSGYSDYKV VGTGGIVGSKGE QQANSFPFT SEQ ID NO: 74 SEQ ID NO: 75SEQ ID NO: 76 RASQGFSGWLA VGTGGTVGSKGE QQATSFPLT SEQ ID NO: 77SEQ ID NO: 78 SEQ ID NO: 79 RASQVISSWLA AASSLQS QQADSFPPT SEQ ID NO: 80SEQ ID NO: 81 SEQ ID NO: 82 RASQVISSWFA LQHNSYPPT SEQ ID NO: 83SEQ ID NO: 84 RASQGSSSWFA SEQ ID NO: 85 RASQGISSWFA SEQ ID NO: 86RAGQVISSWLA SEQ ID NO: 87 RASQGIAGWLA SEQ ID NO: 88 RASQGIRNDLGSEQ ID NO: 89 Exemplary CDRH Sequences CDRH1 CDRH2 CDRH3 SYGMHVIWYDGSNEYYADSVKG DRGYTSSWYPDAFDI SEQ ID NO: 91 SEQ ID NO: 92SEQ ID NO: 93 SYAMH VIWYDGSNKYYADSVKG DRGYSSSWYPDAFDI SEQ ID NO: 94SEQ ID NO: 95 SEQ ID NO: 96 TYSMN VISFDGSLKYYADSVKG ERTTLSGSYFDYSEQ ID NO: 97 SEQ ID NO: 98 SEQ ID NO: 99 SYSMN VISHDGSIKYYADSVKGRIAAAGGFHYYYALDV SEQ ID NO: 100 SEQ ID NO: 101 SEQ ID NO: 102 SFSMNYISSRSSTIYIADSVKG RIAAAGPWGYYYAMDV SEQ ID NO: 103 SEQ ID NO: 104SEQ ID NO: 105 SGGYYWT YISSSSSTRYHADSVKG NRGYYYGMDV SEQ ID NO: 106SEQ ID NO: 107 SEQ ID NO: 108 SGGYYWS YISSRSSTIYYADSVKG NRGFYYGMDVSEQ ID NO: 109 SEQ ID NO: 110 SEQ ID NO: 111 SYFWS YIYYSGNTYYNPSLKSDRGHYYGMDV SEQ ID NO: 112 SEQ ID NO: 113 SEQ ID NO: 114 TYYWSHIHYSGNTYYNPSLKS DRGSYYGSDY SEQ ID NO: 115 SEQ ID NO: 116 SEQ ID NO: 117YIYYSGSTYYNPSLKS DRGYYYGVDV SEQ ID NO: 118 SEQ ID NO: 119YIYYSGSSYYNPSLKS ENTVTIYYNYGMDV SEQ ID NO: 120 SEQ ID NO: 6YIYYSGSTNYNPSLKS SEQ ID NO: 121 LIYTSGSTNYNPSLKS SEQ ID NO: 122LIWYDGSNKYYADSVKG SEQ ID NO: 90

Provided herein are CDR1 regions comprising amino acid residues 23-34 ofSEQ ID NOs: 7 and 11; amino acid residues 24-34 of SEQ ID NOs: 9, 13,15, 17, 19 21, 23, 25, 27 and 29; amino acid residues 23-36 of SEQ IDNOs: 1, 3 and 4; amino acid residues 31-35 of SEQ ID NOs:31, 33, 34, 38,40, 44, 52 and 60 and amino acid residues 31-37 or SEQ ID NOs: 46, 48,50, 54, 56 and 58.

CDR2 regions are provided comprising amino acid residues 50-56 of SEQ IDNOs: 9, 13, 15, 17, 19, 21, 23, 25, 27 and 29; amino acid residues 50-61of SEQ ID NOs: 7 and 11; amino acid residues 52-62 of SEQ ID NO:4; aminoacid residues 50-65 of SEQ ID NOs: 31, 33, 44 and 52; amino acidresidues 50-66 of SEQ ID NOs: 36, 38, 40, 42 and 60; amino acid residues52-58 of SEQ ID NOs: 1 and 3 and amino acid residues 52-67 of SEQ IDNOs: 46, 48, 50, 54, 56 and 58.

CDR3 regions comprising amino acid residues 89-97 of SEQ ID NOs: 13, 15,17, 19, 21, 23, 25, 27 and 29; amino acid residues 91-101 of SEQ ID NOs:1 and 3; amino acid residues 94-106 of SEQ ID NOs: 7, 9 and 11; aminoacid residues 98-107 of SEQ ID NOs: 44 and 52; amino acid residues97-105 of SEQ ID NO: 4; amino acid residues 99-110 of SEQ ID NOs: 34 and36; amino acid residues 99-112 of SEQ ID NO: 112; amino acid residues99-113 of SEQ ID NOs: 31 and 33; amino acid residues 99-114 of SEQ IDNOs: 38, 40 and 42; amino acid residues 100-109 of SEQ ID NOs: 46, 48,54, 56 and 58; and amino acid residues 101-019 of SEQ ID NO; 50; arealso provided.

The CDRs disclosed herein include consensus sequences derived fromgroups of related sequences. As described previously, four groups ofvariable region sequences were identified, a kappa group and threelambda groups. The CDRL1 consensus sequence from the kappa groupconsists of RASQX₁X₂SX₃WX₄A (SEQ ID NO:123) where X₁ is selected from Gor V; X₂ is selected from I, F or S; X₃ is selected from S or G and X₄is selected from F or L. The CDRL1 consensus sequence from lambda group1 consists of TLX₁SGYSDYKVD (SEQ ID NO:124) wherein X₁ is selected fromN or S. The CDRL1 consensus sequences from lambda group 3 consists ofTGSSSNX₁GAGYDVH (SEQ ID NO:125) wherein X₁ is selected from I or T.

The CDRL2 consensus sequence from lambda group 1 consists ofVGTGGX₁VGSKGX₂ (SEQ ID NO: 126) wherein X₁ is selected from I or T andX₂ is selected from D or E. The CDRL2 consensus sequence from lambdagroup 3 consists of GSX₁NRPS (SEQ ID NO:127) wherein X₁ is selected fromN or G.

The CDRL3 consensus sequences include GADHGSGX₁NFVYV (SEQ ID NO:128)wherein X₁ is S or N.

The CDRH1 consensus sequence from the kappa group consists of SGGYYWX₁(SEQ ID NO:129) wherein X₁ is selected from S or T. The CDRH1 consensussequence from lambda group 1 consists of X₁X₂SMN (SEQ ID NO:131) whereinX₁ is selected from S or T and X₂ is selected from Y or F. The CDRH1consensus sequence from lambda group 2 consists of SYX₁MH (SEQ IDNO:130), wherein X₁ is selected from G or A.

The CDRH2 consensus sequence from the kappa group consists ofX₁IX₂YSGX₃X₄YYNPSLKS (SEQ ID NO:132) wherein X₁ is selected from Y or H;X₂ is selected from Y or H; X₃ is selected from S or N and X₄ isselected from T or S. The consensus sequence from lambda group 1consists of YISSX₁SSTX₂YX₃ADSVKG (SEQ ID NO:134) wherein X₁ is selectedfrom R or S, X₂ is selected from I or R, X₃ is selected from I, H or Y.The consensus sequence from lambda group 2 consists ofVISX₁DGSX₂KYYADSVKG (SEQ ID NO:133) wherein X₁ is F or H and X₂ is L orT. The CDRH2 consensus sequence from lambda group 3 consists ofVIWYDGSNX₁YYADSVKG (SEQ ID NO:135) wherein X₁ is selected from K or E.

The CDRH3 consensus sequence from the kappa group consists ofX₁RGX₂YYGMDV (SEQ ID NO:136) wherein X₁ is selected from N or D and X₂is selected from H, Y or F. The CDRH3 consensus sequence from lambdagroup 1 consists of RIAAAGX₁X₂X₃YYYAX₄DV (SEQ ID NO:137) wherein X₁ isselected from G or P; X₂ is selected from F or W; X₃ is selected from Hor G and X₄ is selected from L and M. The CDRH3 consensus sequence fromlambda group 3 consists of DRGYX₁SSWYPDAFDI (SEQ ID NO:138) wherein X₁is selected from S or T.

Monoclonal Antibodies

The antigen binding proteins that are provided include monoclonalantibodies that bind to IL-23. Monoclonal antibodies may be producedusing any technique known in the art, e.g., by immortalizing spleencells harvested from the transgenic animal after completion of theimmunization schedule. The spleen cells can be immortalized using anytechnique known in the art, e.g., by fusing them with myeloma cells toproduce hybridomas. Myeloma cells for use in hybridoma-producing fusionprocedures preferably are non-antibody-producing, have high fusionefficiency, and enzyme deficiencies that render them incapable ofgrowing in certain selective media which support the growth of only thedesired fused cells (hybridomas). Examples of suitable cell lines foruse in mouse fusions include Sp-20, P3-X63/Ag8, P3-X63-Ag8.653, NS1/1.Ag4 1, Sp210-Ag14, FO, NSO/U, MPC-11, MPC11-X45-GTG 1.7 and S194/5XXO Bul;examples of cell lines used in rat fusions include R210.RCY3, Y3-Ag1.2.3, IR983F and 4B210. Other cell lines useful for cell fusions areU-266, GM1500-GRG2, LICR-LON-HMy2 and UC729-6.

In some instances, a hybridoma cell line is produced by immunizing ananimal (e.g., a transgenic animal having human immunoglobulin sequences)with an IL-23 immunogen; harvesting spleen cells from the immunizedanimal; fusing the harvested spleen cells to a myeloma cell line,thereby generating hybridoma cells; establishing hybridoma cell linesfrom the hybridoma cells, and identifying a hybridoma cell line thatproduces an antibody that binds an IL-23 polypeptide while sparingIL-12. Such hybridoma cell lines, and anti-IL-23 monoclonal antibodiesproduced by them, are aspects of the present application.

Monoclonal antibodies secreted by a hybridoma cell line can be purifiedusing any technique known in the art. Hybridomas or mAbs may be furtherscreened to identify mAbs with particular properties, such as theability to inhibit IL-23-induced activity.

Chimeric and Humanized Antibodies

Chimeric and humanized antibodies based upon the foregoing sequences arealso provided. Monoclonal antibodies for use as therapeutic agents maybe modified in various ways prior to use. One example is a chimericantibody, which is an antibody composed of protein segments fromdifferent antibodies that are covalently joined to produce functionalimmunoglobulin light or heavy chains or immunologically functionalportions thereof. Generally, a portion of the heavy chain and/or lightchain is identical with or homologous to a corresponding sequence inantibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is/are identical with or homologous to a corresponding sequencein antibodies derived from another species or belonging to anotherantibody class or subclass. For methods relating to chimeric antibodies,see, for example, U.S. Pat. No. 4,816,567; and Morrison et al., 1985,Proc. Natl. Acad. Sci. USA 81:6851-6855. CDR grafting is described, forexample, in U.S. Pat. Nos. 6,180,370, 5,693,762, 5,693,761, 5,585,089,and 5,530,101.

One useful type of chimeric antibody is a “humanized” antibody.Generally, a humanized antibody is produced from a monoclonal antibodyraised initially in a non-human animal. Certain amino acid residues inthis monoclonal antibody, typically from non-antigen recognizingportions of the antibody, are modified to be homologous to correspondingresidues in a human antibody of corresponding isotype. Humanization canbe performed, for example, using various methods by substituting atleast a portion of a rodent variable region for the correspondingregions of a human antibody (see, e.g., U.S. Pat. No. 5,585,089, and No.5,693,762; Jones et al., 1986, Nature 321:522-525; Riechmann et al.,1988, Nature 332:323-27; Verhoeyen et al., 1988, Science 239:1534-1536),

In certain embodiments, constant regions from species other than humancan be used along with the human variable region(s) to produce hybridantibodies.

Fully Human Antibodies

Fully human antibodies are also provided. Methods are available formaking fully human antibodies specific for a given antigen withoutexposing human beings to the antigen (“fully human antibodies”). Onespecific means provided for implementing the production of fully humanantibodies is the “humanization” of the mouse humoral immune system.Introduction of human immunoglobulin (Ig) loci into mice in which theendogenous Ig genes have been inactivated is one means of producingfully human monoclonal antibodies (mAbs) in mouse, an animal that can beimmunized with any desirable antigen. Using fully human antibodies canminimize the immunogenic and allergic responses that can sometimes becaused by administering mouse or mouse-derivatized mAbs to humans astherapeutic agents.

Fully human antibodies can be produced by immunizing transgenic animals(usually mice) that are capable of producing a repertoire of humanantibodies in the absence of endogenous immunoglobulin production.Antigens for this purpose typically have six or more contiguous aminoacids, and optionally are conjugated to a carrier, such as a hapten.See, e.g., Jakobovits et al., 1993, Proc. Natl. Acad. Sci. USA90:2551-2555; Jakobovits et al., 1993, Nature 362:255-258; andBruggermann et al., 1993, Year in Immunol. 7:33. In one example of sucha method, transgenic animals are produced by incapacitating theendogenous mouse immunoglobulin loci encoding the mouse heavy and lightimmunoglobulin chains therein, and inserting into the mouse genome largefragments of human genome DNA containing loci that encode human heavyand light chain proteins. Partially modified animals, which have lessthan the full complement of human immunoglobulin loci, are thencross-bred to obtain an animal having all of the desired immune systemmodifications. When administered an immunogen, these transgenic animalsproduce antibodies that are immunospecific for the immunogen but havehuman rather than murine amino acid sequences, including the variableregions. For further details of such methods, see, for example, WIPOpatent publications WO96/33735 and WO94/02602. Additional methodsrelating to transgenic mice for making human antibodies are described inU.S. Pat. Nos. 5,545,807; 6,713,610; 6,673,986; 6,162,963; 5,545,807;6,300,129; 6,255,458; 5,877,397; 5,874,299 and 5,545,806; in WIPO patentpublications WO91/10741, WO90/04036, and in EP 546073B1 and EP 546073A1.

The transgenic mice described above contain a human immunoglobulin geneminilocus that encodes unrearranged human heavy ([mu] and [gamma]) and[kappa] light chain immunoglobulin sequences, together with targetedmutations that inactivate the endogenous [mu] and [kappa] chain loci(Lonberg et al., 1994, Nature 368:856-859). Accordingly, the miceexhibit reduced expression of mouse IgM or [kappa] and in response toimmunization, and the introduced human heavy and light chain transgenesundergo class switching and somatic mutation to generate high affinityhuman IgG [kappa] monoclonal antibodies (Lonberg et al., supra.; Lonbergand Huszar, 1995, Intern. Rev. Immunol. 13: 65-93; Harding and Lonberg,1995, Ann. N.Y Acad. Sci. 764:536-546). The preparation of such mice isdescribed in detail in Taylor et al., 1992, Nucleic Acids Research20:6287-6295; Chen et al., 1993, International Immunology 5:647-656;Tuaillon et al., 1994, J. Immunol. 152:2912-2920; Lonberg et al., 1994,Nature 368:856-859; Lonberg, 1994, Handbook of Exp. Pharmacology113:49-101; Taylor et al., 1994, International Immunology 6:579-591;Lonberg and Huszar, 1995, Intern. Rev. Immunol. 13:65-93; Harding andLonberg, 1995, Ann. N.Y Acad. Sci. 764:536-546; Fishwild et al., 1996,Nature Biotechnology 14:845-85. See, further U.S. Pat. No. 5,545,806;No. 5,569,825; No. 5,625,126; No. 5,633,425; No. 5,789,650; No.5,877,397; No. 5,661,016; No. 5,814,318; No. 5,874,299; and No.5,770,429; as well as U.S. Pat. No. 5,545,807; WIPO Publication Nos. WO93/1227; WO 92/22646; and WO 92/03918. Technologies utilized forproducing human antibodies in these transgenic mice are disclosed alsoin WIPO Publication No. WO 98/24893, and Mendez et al., 1997, NatureGenetics 15:146-156. For example, the HCo7 and HCo12 transgenic micestrains can be used to generate anti-IL-23 antibodies.

Using hybridoma technology, antigen-specific human mAbs with the desiredspecificity can be produced and selected from the transgenic mice suchas those described above. Such antibodies may be cloned and expressedusing a suitable vector and host cell, or the antibodies can beharvested from cultured hybridoma cells.

Fully human antibodies can also be derived from phage-display libraries(such as disclosed in Hoogenboom et al., 1991, J. Mol. Biol. 227:381;Marks et al., 1991, J. Mol. Biol. 222:581; WIPO Publication No. WO99/10494). Phage display techniques mimic immune selection through thedisplay of antibody repertoires on the surface of filamentousbacteriophage, and subsequent selection of phage by their binding to anantigen of choice.

Bispecific or Bifunctional Antigen Binding Proteins

A “bispecific,” “dual-specific” or “bifunctional” antigen bindingprotein or antibody is a hybrid antigen binding protein or antibody,respectively, having two different antigen binding sites, such as one ormore CDRs or one or more variable regions as described above. In someinstances they are an artificial hybrid antibody having two differentheavy/light chain pairs and two different binding sites. Multispecificantigen binding protein or “multispecific antibody” is one that targetsmore than one antigen or epitope. Bispecific antigen binding proteinsand antibodies are a species of multispecific antigen binding proteinantibody and may be produced by a variety of methods including, but notlimited to, fusion of hybridomas or linking of Fab′ fragments. See,e.g., Songsivilai and Lachmann, 1990, Clin. Exp. Immunol. 79:315-321;Kostelny et al., 1992, J. Immunol. 148:1547-1553.

Immunological Fragments

Antigen binding proteins also include immunological fragments of anantibody (e.g., a Fab, a Fab′, a F(ab′)₂, or a scFv). A “Fab fragment”is comprised one light chain (the light chain variable region (V_(L))and its corresponding constant domain (C_(L))) and one heavy chain (theheavy chain variable region (V_(H)) and first constant domain (C_(H)1)).The heavy chain of a Fab molecule cannot form a disulfide bond withanother heavy chain molecule. A “Fab′ fragment” contains one light chainand a portion of one heavy chain that also contains the region betweenthe C_(H)1 and C_(H)2 domains, such that an interchain disulfide bondcan be formed between the two heavy chains of two Fab′ fragments to forman F(ab′)₂ molecule. A “F(ab′)₂ fragment” thus is composed of two Fab′fragments that are held together by a disulfide bond between the twoheavy chains. A “Fv fragment” consists of the variable light chainregion and variable heavy chain region of a single arm of an antibody.Single-chain antibodies “scFv” are Fv molecules in which the heavy andlight chain variable regions have been connected by a flexible linker toform a single polypeptide chain, which forms an antigen binding region.Single chain antibodies are discussed in detail in WIPO Publication No.WO 88/01649, U.S. Pat. No. 4,946,778 and No. 5,260,203; Bird, 1988,Science 242:423; Huston et al., 1988, Proc. Natl. Acad. Sci. U.S.A.85:5879; Ward et al., 1989, Nature 334:544, de Graaf et al., 2002,Methods Mol Biol. 178:379-387; Kortt et al., 1997, Prot. Eng. 10:423;Kortt et al., 2001, Biomol. Eng. 18:95-108 and Kriangkum et al., 2001,Biomol. Eng. 18:31-40. A “Fc” region contains two heavy chain fragmentscomprising the C_(H)1 and C_(H)2 domains of an antibody. The two heavychain fragments are held together by two or more disulfide bonds and byhydrophobic interactions of the C_(H)3 domains.

Also included are domain antibodies, immunologically functionalimmunoglobulin fragments containing only the variable region of a heavychain or the variable region of a light chain. In some instances, two ormore V_(H) regions are covalently joined with a peptide linker to createa bivalent domain antibody. The two V_(H) regions of a bivalent domainantibody may target the same or different antigens. Diabodies arebivalent antibodies comprising two polypeptide chains, wherein eachpolypeptide chain comprises V_(H) and V_(L) domains joined by a linkerthat is too short to allow for pairing between two domains on the samechain, thus allowing each domain to pair with a complementary domain onanother polypeptide chain (see, e.g., Holliger et al., Proc. Natl. Acad.Sci. USA 90:6444-48, 1993 and Poljak et al., Structure 2:1121-23, 1994).Similarly, tribodies and tetrabodies are antibodies comprising three andfour polypeptide chains, respectively, and forming three and fourantigen binding sites, respectively, which can be the same or different.Maxibodies comprise bivalent scFvs covalently attached to the Fc regionof IgG₁, (see, e.g., Fredericks et al, 2004, Protein Engineering, Design& Selection, 17:95-106; Powers et al., 2001, Journal of ImmunologicalMethods, 251:123-135; Shu et al., 1993, Proc. Natl. Acad. Sci. USA90:7995-7999; Hayden et al., 1994, Therapeutic Immunology 1:3-15).

Various Other Forms

Also provided are variant forms of the antigen binding proteinsdisclosed above, some of the antigen binding proteins having, forexample, one or more conservative amino acid substitutions in one ormore of the heavy or light chains, variable regions or CDRs listed inTABLES 1 and 2.

Naturally-occurring amino acids may be divided into classes based oncommon side chain properties: hydrophobic (norleucine, Met, Ala, Val,Leu, Ile); neutral hydrophilic (Cys, Ser, Thr, Asn, Gln); acidic (Asp,Glu); basic (His, Lys, Arg); residues that influence chain orientation(Gly, Pro); and aromatic (Trp, Tyr, Phe).

Conservative amino acid substitutions may involve exchange of a memberof one of these classes with another member of the same class.Conservative amino acid substitutions may encompass non-naturallyoccurring amino acid residues, which are typically incorporated bychemical peptide synthesis rather than by synthesis in biologicalsystems. These include peptidomimetics and other reversed or invertedforms of amino acid moieties. Such substantial modifications in thefunctional and/or biochemical characteristics of the antigen bindingproteins described herein may be achieved by creating substitutions inthe amino acid sequence of the heavy and light chains that differsignificantly in their effect on maintaining (a) the structure of themolecular backbone in the area of the substitution, for example, as asheet or helical conformation, (b) the charge or hydrophobicity of themolecule at the target site, or (c) the bulkiness of the side chain.

Non-conservative substitutions may involve the exchange of a member ofone of the above classes for a member from another class. Suchsubstituted residues may be introduced into regions of the antibody thatare homologous with human antibodies, or into the non-homologous regionsof the molecule.

In making such changes, according to certain embodiments, thehydropathic index of amino acids may be considered. The hydropathicprofile of a protein is calculated by assigning each amino acid anumerical value (“hydropathy index”) and then repetitively averagingthese values along the peptide chain. Each amino acid has been assigneda hydropathic index on the basis of its hydrophobicity and chargecharacteristics. They are: isoleucine (+4.5); valine (+4.2); leucine(+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine(+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8);tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2);glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5);lysine (−3.9); and arginine (−4.5).

The importance of the hydropathic profile in conferring interactivebiological function on a protein is understood in the art (see, e.g.,Kyte et al., 1982, J. Mol. Biol. 157:105-131). It is known that certainamino acids may be substituted for other amino acids having a similarhydropathic index or score and still retain a similar biologicalactivity. In making changes based upon the hydropathic index, in certainembodiments, the substitution of amino acids whose hydropathic indicesare within ±2 is included. In some aspects, those which are within ±1are included, and in other aspects, those within ±0.5 are included.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity,particularly where the biologically functional protein or peptidethereby created is intended for use in immunological embodiments, as inthe present case. In certain embodiments, the greatest local averagehydrophilicity of a protein, as governed by the hydrophilicity of itsadjacent amino acids, correlates with its immunogenicity and antigenbinding or immunogenicity, that is, with a biological property of theprotein.

The following hydrophilicity values have been assigned to these aminoacid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1);glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2);glycine (0); threonine (−0.4); proline (−0.5±1); alanine (−0.5);histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5);leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5)and tryptophan (−3.4). In making changes based upon similarhydrophilicity values, in certain embodiments, the substitution of aminoacids whose hydrophilicity values are within ±2 is included, in otherembodiments, those which are within ±1 are included, and in still otherembodiments, those within ±0.5 are included. In some instances, one mayalso identify epitopes from primary amino acid sequences on the basis ofhydrophilicity. These regions are also referred to as “epitopic coreregions.”

Exemplary conservative amino acid substitutions are set forth in TABLE4.

TABLE 4 Conservative Amino Acid Substitutions Residue Sub Residue SubResidue Sub Residue Sub Ala Ser Gln Asn Leu Ile, Val Thr Ser Arg Lys GluAsp Lys Arg, Trp Tyr Gln, Glu Asn Gln, Gly Pro Met Leu, Ile Tyr Trp, HisPhe Asp Glu His Asn, Phe Met, Val Ile, Leu Gln Leu, Tyr Cys Ser Ile Leu,Ser Thr Thr Ser Val Residue = Original Residue Sub = ExemplarySubstitution

A skilled artisan will be able to determine suitable variants ofpolypeptides as set forth herein using well-known techniques. Oneskilled in the art may identify suitable areas of the molecule that maybe changed without destroying activity by targeting regions not believedto be important for activity. The skilled artisan also will be able toidentify residues and portions of the molecules that are conserved amongsimilar polypeptides. In further embodiments, even areas that may beimportant for biological activity or for structure may be subject toconservative amino acid substitutions without destroying the biologicalactivity or without adversely affecting the polypeptide structure.

Additionally, one skilled in the art can review structure-functionstudies identifying residues in similar polypeptides that are importantfor activity or structure. In view of such a comparison, one can predictthe importance of amino acid residues in a protein that correspond toamino acid residues important for activity or structure in similarproteins. One skilled in the art may opt for chemically similar aminoacid substitutions for such predicted important amino acid residues.

One skilled in the art can also analyze the 3-dimensional structure andamino acid sequence in relation to that structure in similarpolypeptides. In view of such information, one skilled in the art maypredict the alignment of amino acid residues of an antibody with respectto its three dimensional structure. One skilled in the art may choosenot to make radical changes to amino acid residues predicted to be onthe surface of the protein, since such residues may be involved inimportant interactions with other molecules. Moreover, one skilled inthe art may generate test variants containing a single amino acidsubstitution at each desired amino acid residue. These variants can thenbe screened using assays for IL-23 activity, (see examples below) thusyielding information regarding which amino acids can be changed andwhich must not be changed. In other words, based on information gatheredfrom such routine experiments, one skilled in the art can readilydetermine the amino acid positions where further substitutions should beavoided either alone or in combination with other mutations.

A number of scientific publications have been devoted to the predictionof secondary structure. See, Moult, 1996, Curr. Op. in Biotech.7:422-427; Chou et al., 1974, Biochem. 13:222-245; Chou et al., 1974,Biochemistry 113:211-222; Chou et al., 1978, Adv. Enzymol. Relat. AreasMol. Biol. 47:45-148; Chou et al., 1979, Ann. Rev. Biochem. 47:251-276;and Chou et al., 1979, Biophys. J. 26:367-384. Moreover, computerprograms are currently available to assist with predicting secondarystructure. One method of predicting secondary structure is based uponhomology modeling. For example, two polypeptides or proteins that have asequence identity of greater than 30%, or similarity greater than 40%often have similar structural topologies. The recent growth of theprotein structural database (PDB) has provided enhanced predictabilityof secondary structure, including the potential number of folds within apolypeptide's or protein's structure. See, Holm et al., 1999, Nucl.Acid. Res. 27:244-247. It has been suggested (Brenner et al., 1997,Curr. Op. Struct. Biol. 7:369-376) that there are a limited number offolds in a given polypeptide or protein and that once a critical numberof structures have been resolved, structural prediction will becomedramatically more accurate.

Additional methods of predicting secondary structure include “threading”(Jones, 1997, Curr. Opin. Struct. Biol. 7:377-387; Sippl et al., 1996,Structure 4:15-19), “profile analysis” (Bowie et al., 1991, Science253:164-170; Gribskov et al., 1990, Meth. Enzym. 183:146-159; Gribskovet al., 1987, Proc. Nat. Acad. Sci. 84:4355-4358), and “evolutionarylinkage” (See, Holm, 1999, supra; and Brenner, 1997, supra).

In some embodiments, amino acid substitutions are made that: (1) reducesusceptibility to proteolysis, (2) reduce susceptibility to oxidation,(3) alter binding affinity for forming protein complexes, (4) alterligand or antigen binding affinities, and/or (4) confer or modify otherphysicochemical or functional properties on such polypeptides, such asmaintaining the structure of the molecular backbone in the area of thesubstitution, for example, as a sheet or helical conformation;maintaining or altering the charge or hydrophobicity of the molecule atthe target site, or maintaining or altering the bulkiness of a sidechain.

For example, single or multiple amino acid substitutions (in certainembodiments, conservative amino acid substitutions) may be made in thenaturally-occurring sequence. Substitutions can be made in that portionof the antibody that lies outside the domain(s) forming intermolecularcontacts). In such embodiments, conservative amino acid substitutionscan be used that do not substantially change the structuralcharacteristics of the parent sequence (e.g., one or more replacementamino acids that do not disrupt the secondary structure thatcharacterizes the parent or native antigen binding protein). Examples ofart-recognized polypeptide secondary and tertiary structures aredescribed in Proteins, Structures and Molecular Principles (Creighton,Ed.), 1984, W. H. New York: Freeman and Company; Introduction to ProteinStructure (Branden and Tooze, eds.), 1991, New York: Garland Publishing;and Thornton et al., 1991, Nature 354:105.

Additional variants include cysteine variants wherein one or morecysteine residues in the parent or native amino acid sequence aredeleted from or substituted with another amino acid (e.g., serine).Cysteine variants are useful, inter alia when antibodies (for example)must be refolded into a biologically active conformation. Cysteinevariants may have fewer cysteine residues than the native protein, andtypically have an even number to minimize interactions resulting fromunpaired cysteines.

The heavy and light chain variable region and CDRs that are disclosedcan be used to prepare antigen binding proteins that contain an antigenbinding region that can specifically bind to an IL-23 polypeptide.“Antigen binding region” means a protein, or a portion of a protein,that specifically binds a specified antigen, such as the region thatcontains the amino acid residues that interact with an antigen andconfer on the antigen binding protein its specificity and affinity forthe target antigen. An antigen binding region may include one or moreCDRs and certain antigen binding regions also include one or more“framework” regions. For example, one or more of the CDRs listed inTABLE 3 can be incorporated into a molecule (e.g., a polypeptide)covalently or noncovalently to make an immunoadhesion. An immunoadhesionmay incorporate the CDR(s) as part of a larger polypeptide chain, maycovalently link the CDR(s) to another polypeptide chain, or mayincorporate the CDR(s) noncovalently. The CDR(s) enable theimmunoadhesion to bind specifically to a particular antigen of interest(e.g., an IL-23 polypeptide).

Other antigen binding proteins include mimetics (e.g., “peptidemimetics” or “peptidomimetics”) based upon the variable regions and CDRsthat are described herein. These analogs can be peptides, non-peptidesor combinations of peptide and non-peptide regions. Fauchere, 1986, Adv.Drug Res. 15:29; Veber and Freidinger, 1985, TINS p. 392; and Evans etal., 1987, J. Med. Chem. 30:1229. Peptide mimetics that are structurallysimilar to therapeutically useful peptides may be used to produce asimilar therapeutic or prophylactic effect. Such compounds are oftendeveloped with the aid of computerized molecular modeling. Generally,peptidomimetics are proteins that are structurally similar to an antigenbinding protein displaying a desired biological activity, such as theability to bind IL-23, but peptidomimetics have one or more peptidelinkages optionally replaced by a linkage selected from, for example:—CH₂NH—, —CH₂S—, —CH₂—CH₂—, —CH—CH— (cis and trans), —COCH₂—,—CH(OH)CH₂—, and —CH₂SO—, by methods well known in the art. Systematicsubstitution of one or more amino acids of a consensus sequence with aD-amino acid of the same type (e.g., D-lysine in place of L-lysine) maybe used in certain embodiments to generate more stable proteins. Inaddition, constrained peptides comprising a consensus sequence or asubstantially identical consensus sequence variation may be generated bymethods known in the art (Rizo and Gierasch, 1992, Ann. Rev. Biochem.61:387), for example, by adding internal cysteine residues capable offorming intramolecular disulfide bridges which cyclize the peptide.

Derivatives of the antigen binding proteins that are described hereinare also provided. The derivatized antigen binding proteins can compriseany molecule or substance that imparts a desired property to the antigenbinding protein or fragment, such as increased half-life in a particularuse. The derivatized antigen binding protein can comprise, for example,a detectable (or labeling) moiety (e.g., a radioactive, colorimetric,antigenic or enzymatic molecule, a detectable bead (such as a magneticor electrodense (e.g., gold) bead), or a molecule that binds to anothermolecule (e.g., biotin or Streptavidin)), a therapeutic or diagnosticmoiety (e.g., a radioactive, cytotoxic, or pharmaceutically activemoiety), or a molecule that increases the suitability of the antigenbinding protein for a particular use (e.g., administration to a subject,such as a human subject, or other in vivo or in vitro uses). Examples ofmolecules that can be used to derivatize an antigen binding proteininclude albumin (e.g., human serum albumin) and polyethylene glycol(PEG). Albumin-linked and PEGylated derivatives of antigen bindingproteins can be prepared using techniques well known in the art. In oneembodiment, the antigen binding protein is conjugated or otherwiselinked to transthyretin (TTR) or a TTR variant. The TTR or TTR variantcan be chemically modified with, for example, a chemical selected fromthe group consisting of dextran, poly(n-vinyl pyrrolidone), polyethyleneglycols, propropylene glycol homopolymers, polypropylene oxide/ethyleneoxide co-polymers, polyoxyethylated polyols and polyvinyl alcohols.

Other derivatives include covalent or aggregative conjugates of IL-23antigen binding proteins with other proteins or polypeptides, such as byexpression of recombinant fusion proteins comprising heterologouspolypeptides fused to the N-terminus or C-terminus of an IL-23 antigenbinding protein. For example, the conjugated peptide may be aheterologous signal (or leader) polypeptide, e.g., the yeastalpha-factor leader, or a peptide such as an epitope tag. IL-23 antigenbinding protein-containing fusion proteins can comprise peptides addedto facilitate purification or identification of the IL-23 antigenbinding protein (e.g., poly-His). An IL-23 antigen binding protein alsocan be linked to the FLAG peptide as described in Hopp et al., 1988,Bio/Technology 6:1204; and U.S. Pat. No. 5,011,912. The FLAG peptide ishighly antigenic and provides an epitope reversibly bound by a specificmonoclonal antibody (mAb), enabling rapid assay and facile purificationof expressed recombinant protein. Reagents useful for preparing fusionproteins in which the FLAG peptide is fused to a given polypeptide arecommercially available (Sigma, St. Louis, Mo.).

Oligomers that contain one or more IL-23 antigen binding proteins may beemployed as IL-23 antagonists. Oligomers may be in the form ofcovalently-linked or non-covalently-linked dimers, trimers, or higheroligomers. Oligomers comprising two or more IL-23 antigen bindingproteins are contemplated for use, with one example being a homodimer.Other oligomers include heterodimers, homotrimers, heterotrimers,homotetramers, heterotetramers, etc. Oligomers comprising multipleIL-23-binding proteins joined via covalent or non-covalent interactionsbetween peptide moieties fused to the IL-23 antigen binding proteins,are also included. Such peptides may be peptide linkers (spacers), orpeptides that have the property of promoting oligomerization. Among thesuitable peptide linkers are those described in U.S. Pat. Nos. 4,751,180and 4,935,233. Leucine zippers and certain polypeptides derived fromantibodies are among the peptides that can promote oligomerization ofIL-23 antigen binding proteins attached thereto. Examples of leucinezipper domains suitable for producing soluble oligomeric proteins aredescribed in WIPO Publication No. WO 94/10308; Hoppe et al., 1994, FEBSLetters 344:191; and Fanslow et al., 1994, Semin. Immunol. 6:267-278. Inone approach, recombinant fusion proteins comprising an IL-23 antigenbinding protein fragment or derivative fused to a leucine zipper peptideare expressed in suitable host cells, and the soluble oligomeric IL-23antigen binding protein fragments or derivatives that form are recoveredfrom the culture supernatant.

Such oligomers may comprise from two to four IL-23 antigen bindingproteins. The IL-23 antigen binding protein moieties of the oligomer maybe in any of the forms described above, e.g., variants or fragments.Preferably, the oligomers comprise IL-23 antigen binding proteins thathave IL-23 binding activity. Oligomers may be prepared usingpolypeptides derived from immunoglobulins. Preparation of fusionproteins comprising certain heterologous polypeptides fused to variousportions of antibody-derived polypeptides (including the Fc domain) hasbeen described, e.g., by Ashkenazi et al., 1991, Proc. Natl. Acad. Sci.USA 88:10535; Byrn et al., 1990, Nature 344:677; and Hollenbaugh et al.,1992 “Construction of Immunoglobulin Fusion Proteins”, in CurrentProtocols in Immunology, Suppl. 4, pages 10.19.1-10.19.11.

Also included are dimers comprising two fusion proteins created byfusing an IL-23 antigen binding protein to the Fc region of an antibody.The dimer can be made by, for example, inserting a gene fusion encodingthe fusion protein into an appropriate expression vector, expressing thegene fusion in host cells transformed with the recombinant expressionvector, and allowing the expressed fusion protein to assemble much likeantibody molecules, whereupon interchain disulfide bonds form betweenthe Fc moieties to yield the dimer. Such Fc polypeptides include nativeand mutein forms of polypeptides derived from the Fc region of anantibody. Truncated forms of such polypeptides containing the hingeregion that promotes dimerization also are included. Fusion proteinscomprising Fc moieties (and oligomers formed therefrom) offer theadvantage of facile purification by affinity chromatography over ProteinA or Protein G columns. One suitable Fc polypeptide, described in WIPOPublication No. WO 93/10151 and U.S. Pat. Nos. 5,426,048 and 5,262,522,is a single chain polypeptide extending from the N-terminal hinge regionto the native C-terminus of the Fc region of a human IgG1 antibody.Another useful Fc polypeptide is the Fc mutein described in U.S. Pat.No. 5,457,035, and in Baum et al., 1994, EMBO J. 13:3992-4001. The aminoacid sequence of this mutein is identical to that of the native Fcsequence presented in WIPO Publication No. WO 93/10151, except thatamino acid 19 has been changed from Leu to Ala, amino acid 20 has beenchanged from Leu to Glu, and amino acid 22 has been changed from Gly toAla. The mutein exhibits reduced affinity for Fc receptors.

Glycosylation

The antigen binding protein may have a glycosylation pattern that isdifferent or altered from that found in the native species. As is knownin the art, glycosylation patterns can depend on both the sequence ofthe protein (e.g., the presence or absence of particular glycosylationamino acid residues, discussed below), or the host cell or organism inwhich the protein is produced. Particular expression systems arediscussed below.

Glycosylation of polypeptides is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tri-peptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tri-peptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-acetylgalactosamine, galactose, or xylose, to a hydroxyamino acid,most commonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition of glycosylation sites to the antigen binding protein isconveniently accomplished by altering the amino acid sequence such thatit contains one or more of the above-described tri-peptide sequences(for N-linked glycosylation sites). The alteration may also be made bythe addition of, or substitution by, one or more serine or threonineresidues to the starting sequence (for O-linked glycosylation sites).For ease, the antigen binding protein amino acid sequence may be alteredthrough changes at the DNA level, particularly by mutating the DNAencoding the target polypeptide at preselected bases such that codonsare generated that will translate into the desired amino acids.

Another means of increasing the number of carbohydrate moieties on theantigen binding protein is by chemical or enzymatic coupling ofglycosides to the protein. These procedures are advantageous in thatthey do not require production of the protein in a host cell that hasglycosylation capabilities for N- and O-linked glycosylation. Dependingon the coupling mode used, the sugar(s) may be attached to (a) arginineand histidine, (b) free carboxyl groups, (c) free sulfhydryl groups suchas those of cysteine, (d) free hydroxyl groups such as those of serine,threonine, or hydroxyproline, (e) aromatic residues such as those ofphenylalanine, tyrosine, or tryptophan, or (f) the amide group ofglutamine. These methods are described in PCT Publication No. WO87/05330, and in Aplin and Wriston, 1981, CRC Crit. Rev, Biochem., pp.259-306.

Removal of carbohydrate moieties present on the starting antigen bindingprotein may be accomplished chemically or enzymatically. Chemicaldeglycosylation requires exposure of the protein to the compoundtrifluoromethanesulfonic acid, or an equivalent compound. This treatmentresults in the cleavage of most or all sugars except the linking sugar(N-acetylglucosamine or N-acetylgalactosamine), while leaving thepolypeptide intact. Chemical deglycosylation is described by Hakimuddinet al., 1987, Arch. Biochem. Biophys. 259:52 and by Edge et al., 1981,Anal. Biochem. 118:131. Enzymatic cleavage of carbohydrate moieties onpolypeptides can be achieved by the use of a variety of endo- andexo-glycosidases as described by Thotakura et al., 1987, Meth. Enzymol.138:350. Glycosylation at potential glycosylation sites may be preventedby the use of the compound tunicamycin as described by Duskin et al.,1982, J. Biol. Chem. 257:3105. Tunicamycin blocks the formation ofprotein-N-glycoside linkages.

Hence, aspects include glycosylation variants of the antigen bindingproteins wherein the number and/or type of glycosylation site(s) hasbeen altered compared to the amino acid sequences of the parentpolypeptide. In certain embodiments, antigen binding protein variantscomprise a greater or a lesser number of N-linked glycosylation sitesthan the parent polypeptide. Substitutions that eliminate or alter thissequence will prevent addition of an N-linked carbohydrate chain presentin the parent polypeptide. For example, the glycosylation can be reducedby the deletion of an Asn or by substituting the Asn with a differentamino acid. Antibodies typically have a N-linked glycosylation site inthe Fc region.

Labels And Effector Groups

Antigen binding proteins may comprise one or more labels. The term“label” or “labeling group” refers to any detectable label. In general,labels fall into a variety of classes, depending on the assay in whichthey are to be detected: a) isotopic labels, which may be radioactive orheavy isotopes; b) magnetic labels (e.g., magnetic particles); c) redoxactive moieties; d) optical dyes; enzymatic groups (e.g. horseradishperoxidase, β-galactosidase, luciferase, alkaline phosphatase); e)biotinylated groups; and f) predetermined polypeptide epitopesrecognized by a secondary reporter (e.g., leucine zipper pair sequences,binding sites for secondary antibodies, metal binding domains, epitopetags, etc.). In some embodiments, the labeling group is coupled to theantigen binding protein via spacer arms of various lengths to reducepotential steric hindrance. Various methods for labeling proteins areknown in the art. Examples of suitable labeling groups include, but arenot limited to, the following: radioisotopes or radionuclides (e.g., ³H,¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I), fluorescent groups (e.g.,FITC, rhodamine, lanthanide phosphors), enzymatic groups (e.g.,horseradish peroxidase, β-galactosidase, luciferase, alkalinephosphatase), chemiluminescent groups, biotinyl groups, or predeterminedpolypeptide epitopes recognized by a secondary reporter (e.g., leucinezipper pair sequences, binding sites for secondary antibodies, metalbinding domains, epitope tags). In some embodiments, the labeling groupis coupled to the antigen binding protein via spacer arms of variouslengths to reduce potential steric hindrance. Various methods forlabeling proteins are known in the art and may be used as is seen fit.

The term “effector group” means any group coupled to an antigen bindingprotein that acts as a cytotoxic agent. Examples for suitable effectorgroups are radioisotopes or radionuclides (e.g., ³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y,⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I). Other suitable groups include toxins,therapeutic groups, or chemotherapeutic groups. Examples of suitablegroups include calicheamicin, auristatins, geldanamycin and maytansine.In some embodiments, the effector group is coupled to the antigenbinding protein via spacer arms of various lengths to reduce potentialsteric hindrance.

Polynucleotides Encoding IL-23 Antigen Binding Proteins

Polynucleotides that encode the antigen binding proteins describedherein, or portions thereof, are also provided, includingpolynucleotides encoding one or both chains of an antibody, or afragment, derivative, mutein, or variant thereof, polynucleotidesencoding heavy chain variable regions or only CDRs, polynucleotidessufficient for use as hybridization probes, PCR primers or sequencingprimers for identifying, analyzing, mutating or amplifying apolynucleotide encoding a polypeptide, anti-sense nucleic acids forinhibiting expression of a polynucleotide, and complementary sequencesof the foregoing. The polynucleotides can be any length. They can be,for example, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 85, 95, 100,125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 750, 1,000, 1,500,3,000, 5,000 or more nucleic acids in length, including all values inbetween, and/or can comprise one or more additional sequences, forexample, regulatory sequences, and/or be part of a largerpolynucleotide, for example, a vector. The polynucleotides can besingle-stranded or double-stranded and can comprise RNA and/or DNAnucleic acids and artificial variants thereof (e.g., peptide nucleicacids).

Polynucleotides encoding certain antigen binding proteins, or portionsthereof (e.g., full length antibody, heavy or light chain, variabledomain, or a CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, or CDRL3) may beisolated from B-cells of mice that have been immunized with IL-23 or animmunogenic fragment thereof. The polynucleotide may be isolated byconventional procedures such as polymerase chain reaction (PCR). Phagedisplay is another example of a known technique whereby derivatives ofantibodies and other antigen binding proteins may be prepared. In oneapproach, polypeptides that are components of an antigen binding proteinof interest are expressed in any suitable recombinant expression system,and the expressed polypeptides are allowed to assemble to form antigenbinding protein molecules. Phage display is also used to derive antigenbinding proteins having different properties (i.e., varying affinitiesfor the antigen to which they bind) via chain shuffling, see Marks etal., 1992, BioTechnology 10:779.

Due to the degeneracy of the genetic code, each of the polypeptidesequences depicted herein are also encoded by a large number of otherpolynucleotide sequences besides those provided. For example, heavychain variable domains provided herein in may be encoded bypolynucleotide sequences SEQ ID NOs: 32, 35, 37, 39, 41, 43, 45, 47, 49,51, 53, 55, 57, or 59. Light chain variable domains may be encoded bypolynucleotide sequences SEQ ID NOs:2, 5, 6, 8, 10, 12, 14, 16, 18, 20,22, 24, 26, or 28. One of ordinary skill in the art will appreciate thatthe present application thus provides adequate written description andenablement for each degenerate nucleotide sequence encoding each antigenbinding protein.

An aspect further provides polynucleotides that hybridize to otherpolynucleotide molecules under particular hybridization conditions.Methods for hybridizing nucleic acids, basic parameters affecting thechoice of hybridization conditions and guidance for devising suitableconditions are well-known in the art. See, e.g., Sambrook, Fritsch, andManiatis (2001, Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y. and Current Protocolsin Molecular Biology, 1995, Ausubel et al., eds., John Wiley & Sons,Inc. As defined herein, a moderately stringent hybridization conditionuses a prewashing solution containing 5× sodium chloride/sodium citrate(SSC), 0.5% SDS, 1.0 mM EDTA (pH 8.0), hybridization buffer of about 50%formamide, 6×SSC, and a hybridization temperature of 55° C. (or othersimilar hybridization solutions, such as one containing about 50%formamide, with a hybridization temperature of 42° C.), and washingconditions of 60° C., in 0.5×SSC, 0.1% SDS. A stringent hybridizationcondition hybridizes in 6×SSC at 45° C., followed by one or more washesin 0.1×SSC, 0.2% SDS at 68° C. Furthermore, one of skill in the art canmanipulate the hybridization and/or washing conditions to increase ordecrease the stringency of hybridization such that polynucleotidescomprising nucleic acid sequences that are at least 65%, 70%, 75%, 80%,85%, 90%, 91%, 92, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to eachother, including all values in between, typically remain hybridized toeach other.

Changes can be introduced by mutation into a polynucleotide, therebyleading to changes in the amino acid sequence of a polypeptide (e.g., anantigen binding protein or antigen binding protein derivative) that itencodes. Mutations can be introduced using any technique known in theart, such as site-directed mutagenesis and random mutagenesis. Mutantpolypeptides can be expressed and selected for a desired property.Mutations can be introduced into a polynucleotide without significantlyaltering the biological activity of a polypeptide that it encodes. Forexample, substitutions at non-essential amino acid residues.Alternatively, one or more mutations can be introduced into apolynucleotide that selectively change the biological activity of apolypeptide that it encodes. For example, the mutation canquantitatively or qualitatively change the biological activity, such asincreasing, reducing or eliminating the activity and changing theantigen specificity of an antigen binding protein.

Another aspect provides polynucleotides that are suitable for use asprimers or hybridization probes for the detection of nucleic acidsequences. A polynucleotide can comprise only a portion of a nucleicacid sequence encoding a full-length polypeptide, for example, afragment that can be used as a probe or primer or a fragment encoding anactive portion (e.g., an IL-23 binding portion) of a polypeptide. Probesbased on the sequence of a nucleic acid can be used to detect thenucleic acid or similar nucleic acids, for example, transcripts encodinga polypeptide. The probe can comprise a label group, e.g., aradioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor.Such probes can be used to identify a cell that expresses thepolypeptide.

Methods of Expressing Antigen Binding Proteins

The antigen binding proteins provided herein may be prepared by any of anumber of conventional techniques. For example, IL-23 antigen bindingproteins may be produced by recombinant expression systems, using anytechnique known in the art. See, e.g., Monoclonal Antibodies,Hybridomas: A New Dimension in Biological Analyses, Kennet et al. (eds.)Plenum Press, New York (1980); and Antibodies: A Laboratory Manual,Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1988).

Expression systems and constructs in the form of plasmids, expressionvectors, transcription or expression cassettes that comprise at leastone polynucleotide as described above are also provided herein, as wellhost cells comprising such expression systems or constructs. As usedherein, “vector” means any molecule or entity (e.g., nucleic acid,plasmid, bacteriophage or virus) suitable for use to transfer proteincoding information into a host cell. Examples of vectors include, butare not limited to, plasmids, viral vectors, non-episomal mammalianvectors and expression vectors, for example, recombinant expressionvectors. Expression vectors, such as recombinant expression vectors, areuseful for transformation of a host cell and contain nucleic acidsequences that direct and/or control (in conjunction with the host cell)expression of one or more heterologous coding regions operatively linkedthereto. An expression construct may include, but is not limited to,sequences that affect or control transcription, translation, and, ifintrons are present, affect RNA splicing of a coding region operablylinked thereto. “Operably linked” means that the components to which theterm is applied are in a relationship that allows them to carry outtheir inherent functions. For example, a control sequence, e.g., apromoter, in a vector that is “operably linked” to a protein codingsequence are arranged such that normal activity of the control sequenceleads to transcription of the protein coding sequence resulting inrecombinant expression of the encoded protein.

Another aspect provides host cells into which an expression vector, suchas a recombinant expression vector, has been introduced. A host cell canbe any prokaryotic cell (for example, E. coli) or eukaryotic cell (forexample, yeast, insect, or mammalian cells (e.g., CHO cells)). VectorDNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. For stabletransfection of mammalian cells, it is known that, depending upon theexpression vector and transfection technique used, only a small fractionof cells may integrate the foreign DNA into their genome. In order toidentify and select these integrants, a gene that encodes a selectablemarker (e.g., for resistance to antibiotics) is generally introducedinto the host cells along with the gene of interest. Preferredselectable markers include those which confer resistance to drugs, suchas G418, hygromycin and methotrexate. Cells stably transfected with theintroduced polynucleotide can be identified by drug selection (e.g.,cells that have incorporated the selectable marker gene will survive,while the other cells die), among other methods.

Antigen binding proteins can be expressed in hybridoma cell lines (e.g.,in particular antibodies may be expressed in hybridomas) or in celllines other than hybridomas. Expression constructs encoding the antigenbinding proteins can be used to transform a mammalian, insect ormicrobial host cell. Transformation can be performed using any knownmethod for introducing polynucleotides into a host cell, including, forexample packaging the polynucleotide in a virus or bacteriophage andtransducing a host cell with the construct by transfection proceduresknown in the art, as exemplified by U.S. Pat. Nos. 4,399,216; 4,912,040;4,740,461; 4,959,455. The optimal transformation procedure used willdepend upon which type of host cell is being transformed. Methods forintroduction of heterologous polynucleotides into mammalian cells arewell known in the art and include, but are not limited to,dextran-mediated transfection, calcium phosphate precipitation,polybrene mediated transfection, protoplast fusion, electroporation,encapsulation of the polynucleotide(s) in liposomes, mixing nucleic acidwith positively-charged lipids, and direct microinjection of the DNAinto nuclei.

Recombinant expression constructs typically comprise a polynucleotideencoding a polypeptide. The polypeptide may comprise one or more of thefollowing: one or more CDRs such as provided herein; a light chainvariable region; a heavy chain variable region; a light chain constantregion; a heavy chain constant region (e.g., C_(H)1, C_(H)2 and/orC_(H)3); and/or another scaffold portion of an IL-23 antigen bindingprotein. These nucleic acid sequences are inserted into an appropriateexpression vector using standard ligation techniques. In one embodiment,the heavy or light chain constant region is appended to the C-terminusof a heavy or light chain variable region provided herein and is ligatedinto an expression vector. The vector is typically selected to befunctional in the particular host cell employed (i.e., the vector iscompatible with the host cell machinery, permitting amplification and/orexpression of the gene can occur). In some embodiments, vectors are usedthat employ protein-fragment complementation assays using proteinreporters, such as dihydrofolate reductase (see, for example, U.S. Pat.No. 6,270,964). Suitable expression vectors can be purchased, forexample, from Invitrogen Life Technologies (Carlsbad, Calif.) or BDBiosciences (San Jose, Calif.). Other useful vectors for cloning andexpressing the antibodies and fragments include those described inBianchi and McGrew, 2003, Biotech. Biotechnol. Bioeng. 84:439-44.Additional suitable expression vectors are discussed, for example, inMethods Enzymol., vol. 185 (D. V. Goeddel, ed.), 1990, New York:Academic Press.

Typically, expression vectors used in any of the host cells will containsequences for plasmid maintenance and for cloning and expression ofexogenous nucleotide sequences. Such sequences, collectively referred toas “flanking sequences” in certain embodiments will typically includeone or more of the following nucleotide sequences: a promoter, one ormore enhancer sequences, an origin of replication, a transcriptionaltermination sequence, a complete intron sequence containing a donor andacceptor splice site, a sequence encoding a leader sequence forpolypeptide secretion, a ribosome binding site, a polyadenylationsequence, a polylinker region for inserting the polynucleotide encodingthe polypeptide to be expressed, and a selectable marker element. Theexpression vectors that are provided may be constructed from a startingvector such as a commercially available vector. Such vectors may or maynot contain all of the desired flanking sequences. Where one or more ofthe flanking sequences described herein are not already present in thevector, they may be individually obtained and ligated into the vector.Methods used for obtaining each of the flanking sequences are well knownto one skilled in the art.

Optionally, the vector may contain a “tag”-encoding sequence, i.e., anoligonucleotide molecule located at the 5′ or 3′ end of the IL-23antigen binding protein coding sequence; the oligonucleotide sequenceencodes polyHis (such as hexaHis), or another “tag” such as FLAG®, HA(hemaglutinin influenza virus), or myc, for which commercially availableantibodies exist. This tag is typically fused to the polypeptide uponexpression of the polypeptide, and can serve as a means for affinitypurification or detection of the IL-23 antigen binding protein from thehost cell. Affinity purification can be accomplished, for example, bycolumn chromatography using antibodies against the tag as an affinitymatrix. Optionally, the tag can subsequently be removed from thepurified IL-23 antigen binding protein by various means such as usingcertain peptidases for cleavage.

Flanking sequences may be homologous (i.e., from the same species and/orstrain as the host cell), heterologous (i.e., from a species other thanthe host cell species or strain), hybrid (i.e., a combination offlanking sequences from more than one source), synthetic or native. Assuch, the source of a flanking sequence may be any prokaryotic oreukaryotic organism, any vertebrate or invertebrate organism, or anyplant, provided that the flanking sequence is functional in, and can beactivated by, the host cell machinery.

Flanking sequences useful in the vectors may be obtained by any ofseveral methods well known in the art. Typically, flanking sequencesuseful herein will have been previously identified by mapping and/or byrestriction endonuclease digestion and can thus be isolated from theproper tissue source using the appropriate restriction endonucleases. Insome cases, the full nucleotide sequence of a flanking sequence may beknown. Here, the flanking sequence may be synthesized using the methodsdescribed herein for nucleic acid synthesis or cloning.

Whether all or only a portion of the flanking sequence is known, it maybe obtained using polymerase chain reaction (PCR) and/or by screening agenomic library with a suitable probe such as an oligonucleotide and/orflanking sequence fragment from the same or another species. Where theflanking sequence is not known, a fragment of DNA containing a flankingsequence may be isolated from a larger piece of DNA that may contain,for example, a coding sequence or even another gene or genes. Isolationmay be accomplished by restriction endonuclease digestion to produce theproper DNA fragment followed by isolation using agarose gelpurification, Qiagen® column chromatography (Qiagen, Chatsworth,Calif.), or other methods known to the skilled artisan. The selection ofsuitable enzymes to accomplish this purpose will be readily apparent toone of ordinary skill in the art.

An origin of replication is typically a part of those prokaryoticexpression vectors purchased commercially, and the origin aids in theamplification of the vector in a host cell. If the vector of choice doesnot contain an origin of replication site, one may be chemicallysynthesized based on a known sequence, and ligated into the vector. Forexample, the origin of replication from the plasmid pBR322 (New EnglandBiolabs, Beverly, Mass.) is suitable for most gram-negative bacteria,and various viral origins (e.g., SV40, polyoma, adenovirus, vesicularstomatitus virus (VSV), or papillomaviruses such as HPV or BPV) areuseful for cloning vectors in mammalian cells. Generally, the origin ofreplication component is not needed for mammalian expression vectors(for example, the SV40 origin is often used only because it alsocontains the virus early promoter).

A transcription termination sequence is typically located 3′ to the endof a polypeptide coding region and serves to terminate transcription.Usually, a transcription termination sequence in prokaryotic cells is aG-C rich fragment followed by a poly-T sequence. While the sequence iseasily cloned from a library or even purchased commercially as part of avector, it can also be readily synthesized using methods for nucleicacid synthesis such as those described herein.

A selectable marker gene encodes a protein necessary for the survivaland growth of a host cell grown in a selective culture medium. Typicalselection marker genes encode proteins that (a) confer resistance toantibiotics or other toxins, e.g., ampicillin, tetracycline, orkanamycin for prokaryotic host cells; (b) complement auxotrophicdeficiencies of the cell; or (c) supply critical nutrients not availablefrom complex or defined media. Specific selectable markers are thekanamycin resistance gene, the ampicillin resistance gene, and thetetracycline resistance gene. Advantageously, a neomycin resistance genemay also be used for selection in both prokaryotic and eukaryotic hostcells.

Other selectable genes may be used to amplify the gene that will beexpressed. Amplification is the process wherein genes that are requiredfor production of a protein critical for growth or cell survival arereiterated in tandem within the chromosomes of successive generations ofrecombinant cells. Examples of suitable selectable markers for mammaliancells include dihydrofolate reductase (DHFR) and promoterless thymidinekinase genes. Mammalian cell transformants are placed under selectionpressure wherein only the transformants are uniquely adapted to surviveby virtue of the selectable gene present in the vector. Selectionpressure is imposed by culturing the transformed cells under conditionsin which the concentration of selection agent in the medium issuccessively increased, thereby leading to the amplification of both theselectable gene and the DNA that encodes another gene, such as anantigen binding protein that binds to IL-23. As a result, increasedquantities of a polypeptide such as an antigen binding protein aresynthesized from the amplified DNA.

A ribosome-binding site is usually necessary for translation initiationof rnRNA and is characterized by a Shine-Dalgarno sequence (prokaryotes)or a Kozak sequence (eukaryotes). The element is typically located 3′ tothe promoter and 5′ to the coding sequence of the polypeptide to beexpressed.

In some cases, such as where glycosylation is desired in a eukaryotichost cell expression system, one may manipulate the various pre- orpro-sequences to improve glycosylation or yield. For example, one mayalter the peptidase cleavage site of a particular signal peptide, or addprosequences, which also may affect glycosylation. The final proteinproduct may have, in the −1 position (relative to the first amino acidof the mature protein), one or more additional amino acids incident toexpression, which may not have been totally removed. For example, thefinal protein product may have one or two amino acid residues found inthe peptidase cleavage site, attached to the amino-terminus.Alternatively, use of some enzyme cleavage sites may result in aslightly truncated form of the desired polypeptide, if the enzyme cutsat such area within the mature polypeptide.

Expression and cloning will typically contain a promoter that isrecognized by the host organism and operably linked to the moleculeencoding an IL-23 antigen binding protein. Promoters are untranscribedsequences located upstream (i.e., 5′) to the start codon of a structuralgene (generally within about 100 to 1000 bp) that control transcriptionof the structural gene. Promoters are conventionally grouped into one oftwo classes: inducible promoters and constitutive promoters. Induciblepromoters initiate increased levels of transcription from DNA undertheir control in response to some change in culture conditions, such asthe presence or absence of a nutrient or a change in temperature.Constitutive promoters, on the other hand, uniformly transcribe a geneto which they are operably linked, that is, with little or no controlover gene expression. A large number of promoters, recognized by avariety of potential host cells, are well known. A suitable promoter isoperably linked to the DNA encoding a heavy chain variable region or alight chain variable region of an IL-23 antigen binding protein byremoving the promoter from the source DNA by restriction enzymedigestion and inserting the desired promoter sequence into the vector.

Suitable promoters for use with yeast hosts are also well known in theart. Yeast enhancers are advantageously used with yeast promoters.Suitable promoters for use with mammalian host cells are well known andinclude, but are not limited to, those obtained from the genomes ofviruses such as polyoma virus, fowlpox virus, adenovirus (such asAdenovirus 2), bovine papilloma virus, avian sarcoma virus,cytomegalovirus, retroviruses, hepatitis-B virus, and Simian Virus 40(SV40). Other suitable mammalian promoters include heterologousmammalian promoters, for example, heat-shock promoters and the actinpromoter.

Additional promoters which may be of interest include, but are notlimited to: SV40 early promoter (Benoist and Chambon, 1981, Nature290:304-310); CMV promoter (Thornsen et al., 1984, Proc. Natl. Acad.U.S.A. 81:659-663); the promoter contained in the 3′ long terminalrepeat of Rous sarcoma virus (Yamamoto et al., 1980, Cell 22:787-797);herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad.Sci. U.S.A. 78:1444-1445); promoter and regulatory sequences from themetallothionine gene (Prinster et al., 1982, Nature 296:39-42); andprokaryotic promoters such as the beta-lactamase promoter(Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. U.S.A.75:3727-3731); or the tac promoter (DeBoer et al., 1983, Proc. Natl.Acad. Sci. U.S.A. 80:21-25). Also of interest are the following animaltranscriptional control regions, which exhibit tissue specificity andhave been utilized in transgenic animals: the elastase I gene controlregion that is active in pancreatic acinar cells (Swift et al., 1984,Cell 38:639-646; Ornitz et al., 1986, Cold Spring Harbor Symp. Quant.Biol. 50:399-409; MacDonald, 1987, Hepatology 7:425-515); the insulingene control region that is active in pancreatic beta cells (Hanahan,1985, Nature 315:115-122); the immunoglobulin gene control region thatis active in lymphoid cells (Grosschedl et al., 1984, Cell 38:647-658;Adames et al., 1985, Nature 318:533-538; Alexander et al., 1987, Mol.Cell. Biol. 7:1436-1444); the mouse mammary tumor virus control regionthat is active in testicular, breast, lymphoid and mast cells (Leder etal., 1986, Cell 45:485-495); the albumin gene control region that isactive in liver (Pinkert et al., 1987, Genes and Devel. 1:268-276); thealpha-feto-protein gene control region that is active in liver (Krumlaufet al., 1985, Mol. Cell. Biol. 5:1639-1648; Hammer et al., 1987, Science253:53-58); the alpha 1-antitrypsin gene control region that is activein liver (Kelsey et al., 1987, Genes and Devel. 1:161-171); thebeta-globin gene control region that is active in myeloid cells (Mogramet al., 1985, Nature 315:338-340; Kollias et al., 1986, Cell 46:89-94);the myelin basic protein gene control region that is active inoligodendrocyte cells in the brain (Readhead et al., 1987, Cell48:703-712); the myosin light chain-2 gene control region that is activein skeletal muscle (Sani, 1985, Nature 314:283-286); and thegonadotropic releasing hormone gene control region that is active in thehypothalamus (Mason et al., 1986, Science 234:1372-1378).

An enhancer sequence may be inserted into the vector to increasetranscription by higher eukaryotes. Enhancers are cis-acting elements ofDNA, usually about 10-300 bp in length, that act on the promoter toincrease transcription. Enhancers are relatively orientation andposition independent, having been found at positions both 5′ and 3′ tothe transcription unit. Several enhancer sequences available frommammalian genes are known (e.g., globin, elastase, albumin,alpha-feto-protein and insulin). Typically, however, an enhancer from avirus is used. The SV40 enhancer, the cytomegalovirus early promoterenhancer, the polyoma enhancer, and adenovirus enhancers known in theart are exemplary enhancing elements for the activation of eukaryoticpromoters. While an enhancer may be positioned in the vector either 5′or 3′ to a coding sequence, it is typically located at a site 5′ fromthe promoter. A sequence encoding an appropriate native or heterologoussignal sequence (leader sequence or signal peptide) can be incorporatedinto an expression vector, to promote extracellular secretion of theantibody. The choice of signal peptide or leader depends on the type ofhost cells in which the antibody is to be produced, and a heterologoussignal sequence can replace the native signal sequence. Examples ofsignal peptides that are functional in mammalian host cells include thefollowing: the signal sequence for interleukin-7 described in U.S. Pat.No. 4,965,195; the signal sequence for interleukin-2 receptor describedin Cosman et al., 1984, Nature 312:768; the interleukin-4 receptorsignal peptide described in EP Patent No. 0367 566; the type Iinterleukin-1 receptor signal peptide described in U.S. Pat. No.4,968,607; the type II interleukin-1 receptor signal peptide describedin EP Patent No. 0 460 846.

After the vector has been constructed, the completed vector may beinserted into a suitable host cell for amplification and/or polypeptideexpression. The transformation of an expression vector for an antigenbinding protein into a selected host cell may be accomplished by wellknown methods including transfection, infection, calcium phosphateco-precipitation, electroporation, microinjection, lipofection,DEAE-dextran mediated transfection, or other known techniques. Themethod selected will in part be a function of the type of host cell tobe used. These methods and other suitable methods are well known to theskilled artisan, and are set forth, for example, in Sambrook et al.,Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (2001).

A host cell, when cultured under appropriate conditions, synthesizesprotein that can be subsequently collected from the culture medium (ifthe host cell secretes it into the medium) or directly from the hostcell producing it (if it is not secreted). The selection of anappropriate host cell will depend upon various factors, such as desiredexpression levels, polypeptide modifications that are desirable ornecessary for activity (such as glycosylation or phosphorylation) andease of folding into a biologically active molecule.

Mammalian cell lines available as hosts for expression are well known inthe art and include, but are not limited to, immortalized cell linesavailable from the American Type Culture Collection (ATCC), includingbut not limited to Chinese hamster ovary (CHO) cells, HeLa cells, babyhamster kidney (BHK) cells, monkey kidney cells (COS), humanhepatocellular carcinoma cells (e.g., Hep G2), and a number of othercell lines. In certain embodiments, cell lines may be selected throughdetermining which cell lines have high expression levels andconstitutively produce antigen binding proteins with IL-23 bindingproperties. In another embodiment, a cell line from the B cell lineagethat does not make its own antibody but has a capacity to make andsecrete a heterologous antibody can be also selected.

Use of Human IL-23 Antigen Binding Proteins for Diagnostic andTherapeutic Purposes

Antigen binding proteins are useful for detecting IL-23 in biologicalsamples and identification of cells or tissues that produce IL-23.Antigen binding proteins that specifically bind to IL-23 may be used indiagnosis and/or treatment of diseases related to IL-23 in a patient inneed thereof. For one, the IL-23 antigen binding proteins can be used indiagnostic assays, e.g., binding assays to detect and/or quantify IL-23expressed in blood, serum, cells or tissue. In addition, IL-23 antigenbinding proteins can be used to reduce, inhibit, interfere with ormodulate one or more biological activities of IL-23 in a cell or tissue.Thus antigen binding proteins that bind to IL-23 may have therapeuticuse in ameliorating diseases related to IL-23.

Indications

The present invention also relates to the use of IL-23 antigen bindingproteins for use in the prevention or therapeutic treatment of medicaldisorders, such as those disclosed herein. The IL-23 antigen bindingproteins are useful to treat a variety of conditions in which IL-23 isassociated with or plays a role in contributing to the underlyingdisease or disorder or otherwise contributes to a negative symptom.

Conditions effectively treated by IL-23 antigen binding proteins play arole in the inflammatory response. Such inflammatory disorders includeperiodontal disease; lung disorders such as asthma; skin disorders suchas psoriasis, atopic dermatitis, contact dermatitis; rheumatic disorderssuch as rheumatoid arthritis, progressive systemic sclerosis(scleroderma); systemic lupus erythematosus; spondyloarthritis includingankylosing spondylitis, psoriatic arthritis, enteropathic arthritis andreactive arthritis. Also contemplated is uveitis includingVogt-Koyanagi-Harada disease, idiopathic anterior and posterior uveitis,and uveitis associated with spondyloarthritis. Use of IL-23 antigenbinding proteins is also contemplated for the treatment of autoimmunedisorders including multiple sclerosis; autoimmune myocarditis; type 1diabetes and autoimmune thyroiditis.

Degenerative conditions of the gastrointestinal system are treatable orpreventable with IL-23 antigen binding proteins. Such gastrointestinaldisorders including inflammatory bowel disease: Crohn's disease,ulcerative colitis and Celiac disease.

Also included are use of IL-23 antigen binding proteins in treatmentsfor graft-versus-host disease, and complications such as graftrejection, resulting from solid organ transplantation, such as heart,liver, skin, kidney, lung or other transplants, including bone marrowtransplants.

Also provided herein are methods for using IL-23 antigen bindingproteins to treat various oncologic disorders including various forms ofcancer including colon, stomach, prostate, renal cell, cervical andovarian cancers, and lung cancer (SCLC and NSCLC). Also included aresolid tumors, including sarcoma, osteosarcoma, and carcinoma, such asadenocarcinoma and squamous cell carcinoma, esophogeal cancer, gastriccancer, gall bladder carcinoma, leukemia, including acute myelogenousleukemia, chronic myelogenous leukemia, myeloid leukemia, chronic oracute lymphoblastic leukemia and hairy cell leukemia, and multiplemyeloma.

Diagnostic Methods

The antigen binding proteins of the described can be used for diagnosticpurposes to detect, diagnose, or monitor diseases and/or conditionsassociated with IL-23. Examples of methods useful in the detection ofthe presence of IL-23 include immunoassays, such as the enzyme linkedimmunosorbant assay (ELISA) and the radioimmunoassay (RIA).

For diagnostic applications, the antigen binding protein typically willbe labeled with a detectable labeling group. Suitable labeling groupsinclude, but are not limited to, the following: radioisotopes orradionuclides (e.g., ³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I),fluorescent groups (e.g., FITC, rhodamine, lanthanide phosphors),enzymatic groups (e.g., horseradish peroxidase, β-galactosidase,luciferase, alkaline phosphatase), chemiluminescent groups, biotinylgroups, or predetermined polypeptide epitopes recognized by a secondaryreporter (e.g., leucine zipper pair sequences, binding sites forsecondary antibodies, metal binding domains, epitope tags). In someembodiments, the labelling group is coupled to the antigen bindingprotein via spacer arms of various lengths to reduce potential sterichindrance. Various methods for labelling proteins are known in the artand may be used.

Other diagnostic methods are provided for identifying a cell or cellsthat express IL-23. In a specific embodiment, the antigen bindingprotein is labeled with a labeling group and the binding of the labeledantigen binding protein to IL-23 is detected. In a further specificembodiment, the binding of the antigen binding protein to IL-23 isdetected in vivo. In a further specific embodiment, the IL-23 antigenbinding protein is isolated and measured using techniques known in theart. See, for example, Harlow and Lane, 1988, Antibodies: A LaboratoryManual, New York: Cold Spring Harbor (ed. 1991 and periodicsupplements); John E. Coligan, ed., 1993, Current Protocols InImmunology New York: John Wiley & Sons.

Other methods provide for detecting the presence of a test molecule thatcompetes for binding to IL-23 with the antigen binding proteinsprovided. An example of one such assay would involve detecting theamount of free antigen binding protein in a solution containing anamount of IL-23 in the presence or absence of the test molecule. Anincrease in the amount of free antigen binding protein (i.e., theantigen binding protein not bound to IL-23) would indicate that the testmolecule is capable of competing for IL-23 binding with the antigenbinding protein. In one embodiment, the antigen binding protein islabeled with a labeling group. Alternatively, the test molecule islabeled and the amount of free test molecule is monitored in thepresence and absence of an antigen binding protein.

Methods of Treatment: Pharmaceutical Formulations, Routes ofAdministration

Pharmaceutical compositions that comprise a therapeutically effectiveamount of one or a plurality of the antigen binding proteins and apharmaceutically acceptable excipient, diluent, carrier, solubilizer,emulsifier, preservative, and/or adjuvant are provided. In addition,methods of treating a patient by administering such pharmaceuticalcomposition are included. The term “patient” includes human patients.The terms “treat” and “treatment” encompass alleviation or prevention ofat least one symptom or other aspect of a disorder, or reduction ofdisease severity, and the like. The term “therapeutically effectiveamount” or “effective amount” refers to the amount of an IL-23 antigenbinding protein determined to produce any therapeutic response in amammal. Such therapeutically effective amounts are readily ascertainedby one of ordinary skill in the art.

An antigen binding protein need not affect a complete cure, or eradicateevery symptom or manifestation of a disease, to constitute a viabletherapeutic agent. As is recognized in the pertinent field, drugsemployed as therapeutic agents may reduce the severity of a givendisease state, but need not abolish every manifestation of the diseaseto be regarded as useful therapeutic agents. Similarly, aprophylactically administered treatment need not be completely effectivein preventing the onset of a condition in order to constitute a viableprophylactic agent. Simply reducing the impact of a disease (forexample, by reducing the number or severity of its symptoms, or byincreasing the effectiveness of another treatment, or by producinganother beneficial effect), or reducing the likelihood that the diseasewill occur or worsen in a subject, is sufficient. Certain methodsprovided herein comprise administering to a patient an IL-23 antagonist(such as the antigen binding proteins disclosed herein) in an amount andfor a time sufficient to induce a sustained improvement over baseline ofan indicator that reflects the severity of the particular disorder.

As is understood in the pertinent field, pharmaceutical compositionscomprising the molecules of the invention are administered to a patientin a manner appropriate to the indication. Pharmaceutical compositionsmay be administered by any suitable technique, including but not limitedto, parenterally, topically, or by inhalation. If injected, thepharmaceutical composition can be administered, for example, viaintra-articular, intravenous, intramuscular, intralesional,intraperitoneal or subcutaneous routes, by bolus injection, orcontinuous infusion. Localized administration, e.g. at a site of diseaseor injury is contemplated, as are transdermal delivery and sustainedrelease from implants. Delivery by inhalation includes, for example,nasal or oral inhalation, use of a nebulizer, inhalation of theantagonist in aerosol form, and the like. Other alternatives includeeyedrops; oral preparations including pills, syrups, lozenges or chewinggum; and topical preparations such as lotions, gels, sprays, andointments.

Use of antigen binding proteins in ex vivo procedures also iscontemplated. For example, a patient's blood or other bodily fluid maybe contacted with an antigen binding protein that binds IL-23 ex vivo.The antigen binding protein may be bound to a suitable insoluble matrixor solid support material.

Advantageously, antigen binding proteins are administered in the form ofa composition comprising one or more additional components such as aphysiologically acceptable carrier, excipient or diluent. Optionally,the composition additionally comprises one or more physiologicallyactive agents for combination therapy. A pharmaceutical composition maycomprise an IL-23 antigen binding protein together with one or moresubstances selected from the group consisting of a buffer, anantioxidant such as ascorbic acid, a low molecular weight polypeptide(such as those having fewer than 10 amino acids), a protein, an aminoacid, a carbohydrate such as glucose, sucrose or dextrins, a chelatingagent such as EDTA, glutathione, a stabilizer, and an excipient. Neutralbuffered saline or saline mixed with conspecific serum albumin areexamples of appropriate diluents. In accordance with appropriateindustry standards, preservatives such as benzyl alcohol may also beadded. The composition may be formulated as a lyophilizate usingappropriate excipient solutions (e.g., sucrose) as diluents. Suitablecomponents are nontoxic to recipients at the dosages and concentrationsemployed. Further examples of components that may be employed inpharmaceutical formulations are presented in any Remington'sPharmaceutical Sciences including the 21^(st) Ed. (2005), MackPublishing Company, Easton, Pa.

Kits for use by medical practitioners include an IL-23 antigen bindingprotein and a label or other instructions for use in treating any of theconditions discussed herein. In one embodiment, the kit includes asterile preparation of one or more IL-23 binding antigen bindingproteins, which may be in the form of a composition as disclosed above,and may be in one or more vials.

Dosages and the frequency of administration may vary according to suchfactors as the route of administration, the particular antigen bindingproteins employed, the nature and severity of the disease to be treated,whether the condition is acute or chronic, and the size and generalcondition of the subject. Appropriate dosages can be determined byprocedures known in the pertinent art, e.g. in clinical trials that mayinvolve dose escalation studies.

A typical dosage may range from about 0.1 μg/kg to up to about 30 mg/kgor more, depending on the factors mentioned above. In specificembodiments, the dosage may range from 0.1 μg/kg up to about 30 mg/kg,optionally from 1 μg/kg up to about 30 mg/kg, optionally from 10 μg/kgup to about 10 mg/kg, optionally from about 0.1 mg/kg to 5 mg/kg, oroptionally from about 0.3 mg/kg to 3 mg/kg.

Dosing frequency will depend upon the pharmacokinetic parameters of theparticular human IL-23 antigen binding protein in the formulation used.Typically, a clinician administers the composition until a dosage isreached that achieves the desired effect. The composition may thereforebe administered as a single dose, or as two or more doses (which may ormay not contain the same amount of the desired molecule) over time, oras a continuous infusion via an implantation device or catheter.Appropriate dosages may be ascertained through use of appropriatedose-response data. An IL-23 antigen binding protein of the inventionmay be administered, for example, once or more than once, e.g., atregular intervals over a period of time. In particular embodiments, anIL-23 antigen binding protein is administered over a period of at leasta month or more, e.g., for one, two, or three months or evenindefinitely. For treating chronic conditions, long-term treatment isgenerally most effective. However, for treating acute conditions,administration for shorter periods, e.g. from one to six weeks, may besufficient. In general, the antigen binding protein is administereduntil the patient manifests a medically relevant degree of improvementover baseline for the chosen indicator or indicators.

It is contemplated that an IL-23 antigen binding protein be administeredto the patient in an amount and for a time sufficient to induce animprovement, preferably a sustained improvement, in at least oneindicator that reflects the severity of the disorder that is beingtreated. Various indicators that reflect the extent of the patient'sillness, disease or condition may be assessed for determining whetherthe amount and time of the treatment is sufficient. Such indicatorsinclude, for example, clinically recognized indicators of diseaseseverity, symptoms, or manifestations of the disorder in question. Inone embodiment, an improvement is considered to be sustained if thesubject exhibits the improvement on at least two occasions separated bytwo to four weeks. The degree of improvement generally is determined bya physician, who may make this determination based on signs, symptoms,biopsies, or other test results, and who may also employ questionnairesthat are administered to the subject, such as quality-of-lifequestionnaires developed for a given disease.

Particular embodiments of methods and compositions of the inventioninvolve the use of an IL-23 antigen binding protein and one or moreadditional IL-23 antagonists, for example, two or more antigen bindingproteins of the invention, or an antigen binding protein of theinvention and one or more other IL-23 antagonists. Also provided areIL-23 antigen binding proteins administered alone or in combination withother agents useful for treating the condition with which the patient isafflicted. Examples of such agents include both proteinaceous andnon-proteinaceous drugs. Such agents include therapeutic moieties havinganti-inflammatory properties (for example, non-steroidalanti-inflammatory agents, steroids, immunomodulators and/or othercytokine inhibitors such as those that antagonize, for example, IFN-γ,GM-CSF, IL-6, IL-8, IL-17, IL-22 and TNFs), or of an IL-23 antigenbinding protein and one or more other treatments (e.g., surgery,ultrasound, or treatment effective to reduce inflammation). Whenmultiple therapeutics are co-administered, dosages may be adjustedaccordingly, as is recognized or known in the pertinent art. Usefulagents that may be combined with IL-23 antigen binding proteins includethose used to treat, for example, Crohn's disease or ulcerative colitis,such as aminosalicylate (for example, mesalamine), corticosteroids(including prednisone), antibiotics such as metronidazole orciprofloxacin (or other antibiotics useful for treating, for example,patients afflicted with fistulas), and immunosuppressives such asazathioprine, 6-mercaptopurine, methotrexate, tacrolimus andcyclosporine. Such agent(s) may be administered orally or by anotherroute, for example via suppository or enema. Agents which may becombined with IL-23 binding proteins in treatment of psoriasis includecorticosteroids, calcipotriene and other vitamin D derivatives,acetretin and other retinoic acid derivatives, methotrexate, tacrolimus,and cyclosporine used topically or systemically. Such agents can beadministered simultaneously, consecutively, alternately, or according toany other regimen that allows the total course of therapy to beeffective.

In addition to human patients, IL-23 antigen binding proteins are usefulin the treatment of non-human animals, such as domestic pets (dogs,cats, birds, primates, etc.), domestic farm animals (horses cattle,sheep, pigs, birds, etc). In such instances, an appropriate dose may bedetermined according to the animal's body weight. For example, a dose of0.2-1 mg/kg may be used. Alternatively, the dose is determined accordingto the animal's surface area, an exemplary dose ranging from 0.1-20mg/m2, or more preferably, from 5-12 mg/m2. For small animals, such asdogs or cats, a suitable dose is 0.4 mg/kg. IL-23 antigen bindingprotein (preferably constructed from genes derived from the recipientspecies) is administered by injection or other suitable route one ormore times per week until the animal's condition is improved, or it maybe administered indefinitely.

The following examples, including the experiments conducted and theresults achieved, are provided for illustrative purposes only and arenot to be construed as limiting the scope of the appended claims.

EXAMPLES Example 1 Generation of Human IL-23 Antibodies

XenoMouse™ technology (Amgen, Thousand Oaks, Calif.) was used to develophuman monoclonal antibodies that recognize and inhibit native humanIL-23 activity while sparing human IL-12. The antibodies also recognizeand inhibit recombinant cynomologous IL-23 but do not recognize murineor rat IL-23.

Antibodies were selected for recognition and complete inhibition ofnative human IL-23 obtained from human monocyte-derived dendritic cells(MoDCs), using the STAT-luciferase reporter assay described below. Humanmonocytes were isolated from peripheral blood mononuclear cells fromhealthy donors using negative selection (Monocyte Isolation Kit II,Miltenyi Biotec, Auburn, Calif.). MoDCs were generated by culturingmonocytes with human GM-CSF (50 ng/ml) and human IL-4 (100 ng/ml) for 7days in RPMI 1640 with 10% fetal bovine serum complete medium. MoDCswere then washed twice with PBS followed by stimulation with human CD40L(1 μg/ml) for an additional 48 hours. CD40L-stimulated MoDC supernatantcontains IL-23, IL-12 and IL-12/23p40. ELISAs are used to determine theamount of IL-12p70 (R&D System, Minneapolis, Minn.), IL-23(eBiosciences, San Diego, Calif.) and IL-12123p40 (R&D Systems). TheSTAT-luciferase assay responds to IL-23 and not to IL-12 or to freeIL-12/23p40, therefore the assay could be used with crude supernatantsto assess IL-23 activity. For use in the NK cell assay, described below,the native human IL-23 crude supernatant was purified using an IL-23affinity column followed by size exclusion chromatography. Concentrationwas determined using an IL-23 specific ELISA (eBiosciences).

The purified antibody supernatants were also tested against recombinanthuman (rhu) IL-23 and recombinant cynomolgous (cyno) IL-23 in theSTAT-luciferase assay. Of the antibodies tested that completelyinhibited recombinant human IL-23, only half of those antibodiesrecognized and completely inhibited native human IL-23. Recognition andcomplete inhibition of recombinant human IL-23 was not predictive of,nor correlated to, recognition and complete inhibition of native humanIL-23. As shown in FIGS. 1A and 1B, of the antibody supernatants thatcompletely inhibited recombinant human IL-23, only half of thoseantibodies completely inhibited native human IL-23. Those antibodiesthat recognized and completely inhibited native human IL-23 wereselected for further characterization.

Example 2 Functional Assays

a) STAT-Luciferase Assay

It is known that IL-23 binds its heterodimeric receptor and signalsthrough JAK2 and Tyk2 to activate STAT 1, 3, 4 and 5. In this assay,cells transfected with a STAT/luciferase reporter gene are used toassess the ability of the IL-23 antibodies to inhibit IL-23-inducedbioactivity.

Chinese hamster ovary cells expressing human IL-23 receptor aretransiently transfected with STAT-luciferase reporter overnight. IL-23antibodies are serially diluted (12 points of 1:4 serial dilutionsstarting at 37.5 μg/ml) into 96 well plates. Native human IL-23(preparation method is described in Example 1) is added to each well ata concentration of 2 ng/ml and incubated at room temperature for 15-20minutes. The transiently transfected cells are added (8×10³ cells) to afinal volume of 100 μl/well and incubated for 5 hours at 37° C., 10%CO₂. Following incubation, cells are lysed using 100 μL/well Glo Lysisbuffer (1×) (Promega, Madison, Wis.) at room temperature for 5 minutes.Fifty microliters of cell lysate is added to a 96 well plate along with50 μL Bright-Glo luciferase substrate (Promega) and read on aluminometer.

Statistical analysis can be performed using GraphPad PRISM software(GraphPad Software, La Jolla, Calif.). Results can be expressed as themean±standard deviation (SD).

As seen in TABLE 5, all IL-23 antibodies potently and completelyinhibited native human IL-23-induced STAT/luciferase reporter in a dosedependent manner. The antibodies also potently and completely inhibitedrecombinant human (rhu) IL-23 and recombinant cyno (cyno) IL-23. Theantibodies all had IC₅₀ values in the picomolar range.

TABLE 5 Table of mean IC₅₀ (pM) values for IL-23 antibodies in theSTAT-luciferase assay. Native hulL-23 rhuIL-23 Cyno IL-23 antibody IC₅₀+/− SD Repeats IC₅₀ +/− SD Repeats IC₅₀+/− SD Repeats A 114 +/− 70 3 190+/− 99 3  379 +/− 213 3 B 45 +/− 5 4 100 +/− 59 4 130 +/− 60 3 C 107 +/−31 3 211 +/− 93 3 376 +/− 89 3 D 65 +/− 5 3 107 +/− 30 3 184 +/− 77 3 E140 +/− 52 3 142 +/− 52 3 188 +/− 59 3 F  86 +/− 47 4  187 +/− 116 4 366 +/− 219 4 G 156 +/− 74 5  296 +/− 133 5  421 +/− 174 5 H 192 +/− 354  253 +/− 184 4 1024 +/− 533 4 I 208 +/− 33 3  338 +/− 140 3 650 +/− 423 J  83 +/− 54 2 36 +/− 6 2 56 +/− 2 2 K  71 +/− 38 3  43 +/− 20 3  61+/− 10 3 L 113 +/− 80 3 23 +/− 7 3 47 +/− 1 3 M  34 +/− 11 2 40 +/− 8 256 +/− 6 2 N  361 +/− 164 3 145 1 238 1

b) NK Cell Assay

It is known that IL-23 acts on natural killer cells to induce expressionof pro-inflammatory cytokines, such as interferon γ (IFNγ). In thisassay, human primary natural killer (NK) cells are used to assess theability of the IL-23 antibodies to inhibit IL-23-induced IFNγ activityin cells expressing the native receptor for human IL-23.

NK cells are isolated from multiple human donors via negative selection(NK Cell Isolation Kit, Miltenyi Biotec, Auburn, Calif.). Purified NKcells (1×10⁶ cells/ml) are added to 6 well plates in RPMI 1640 plus 10%fetal bovine serum complete medium supplemented with recombinant humanIL-2 (10 ng/ml, R&D Systems, Minneapolis, Minn.), to a final volume of10 ml/well. Cells are cultured for 7 days at 37° C., 5% CO₂. TheIL-2-activated NK cells are then stimulated with rhuIL-23 or cyno IL-23(10 ng/ml) and recombinant human IL-18 (20 ng/ml, R&D Systems,Minneapolis, Minn.) in the presence of serial dilutions (11 points of1:3 serial dilutions starting at 3 μg/ml) of IL-23 antibodies for 24hours. IFNγ levels are measured in the supernatant by IFNγ ELISA (R&DSystems, Minneapolis, Minn.) according to manufacturer's instructions.

Statistical analysis can be performed using GraphPad PRISM software.Results can be expressed as the mean±standard deviation (SD).

As seen in TABLE 6, all antibodies potently inhibited rhuIL-23 and cynoIL-23-induced IFNγ expression in NK cells in a dose dependent manner.The antibodies all had IC₅₀ values in the picomolar range. The assay wasperformed on a subset of antibodies using native human IL-23 (30 μg/ml,preparation method is described in Example 1) and rhuIL-18 (40 ng/ml,R&D Systems) and yielded the results shown in TABLE 6. Consistent withthe selection for IL-23 specific antibodies, these anti-IL-23 antibodieshad no effect on IL-12 stimulated IFNγ production in NK cells using theassay described above, whereas an IL-12p35 specific neutralizingantibody, mAb219 (R&D Systems, Minneapolis, Minn.) potently inhibitedrecombinant human IL-12.

TABLE 6 Table of mean IC₅₀ (pM) values for IL-23 antibodies in the NKcell assay. Native huIL-23 rhuIL-23 Cyno IL-23 antibody IC₅₀ +/− SDRepeats IC₅₀ +/− SD Repeats IC₅₀+/− SD Repeats A 42 +/− 12 2 31 +/− 21 2B  85 +/− 30 2 48 +/− 30 3 19 +/− 8  2 C 32 +/− 19 4 29 +/− 16 2 D 37+/− 21 2 29 +/− 19 2 E 158 +/− 50 2 57 +/− 14 3 21 +/− 3  2 F 25 +/− 152 21 +/− 17 2 G 152 +/− 72 2 45 +/− 30 3 23 +/− 8  2 H 29 +/− 28 2 33+/− 17 2 I 69 1 52 1 J 4 +/− 3 2 5 +/− 3 2 K 7 +/− 2 2 8 +/− 6 2 L 3 +/−1 2 4 +/− 1 2 M  8 1 12 1

c) Human Whole Blood Assay

Human whole blood is collected from multiple healthy donors usingRefludan® (Bayer Pittsburgh, Pa.) as an anti-coagulant. The finalconcentration of Refludan® in whole blood is 10 μg/ml. A stimulationmixture of rhuIL-23 or cyno IL-23 (final concentration 1 ng/ml)+rhuIL-18(final concentration 20 ng/ml)+rhuIL-2 (final concentration 5 ng/ml) inRPMI 1640+10% FBS, is added to a 96 well plate, final volume 20 μl/well.Serially diluted IL-23 antibodies (11 points of 1:3 serial dilutionsstarting from 3 μg/ml) are added at 20 μl/well and incubated with thestimulation mixture for 30 minutes at room temperature. Whole blood isthen added (120 μl/well) and the final volume adjusted to 200 μl/wellwith RPMI 1640+10% FBS. The final concentration of whole blood is 60%.The plates are incubated for 24 hours at 37° C., 5% CO₂. Cell freesupernatants are harvested and IFNγ levels are measured from thesupernatants by IFNγ ELISA (R&D Systems) according to manufacturer'sinstructions.

Statistical analysis can be performed using GraphPad PRISM software.Results can be expressed as the mean±standard deviation (SD).

As seen in TABLE 7, all antibodies potently inhibited rhuIL-23-inducedand cyno-IL-23-induced IFNγ expression in whole blood cells in a dosedependent manner. The antibodies all had IC50 values in the picomolarrange.

TABLE 7 Table of mean IC₅₀ (pM) values for IL-23 antibodies in the IFNγhuman whole blood assay rhuIL-23 Cyno IL-23 antibody IC₅₀ +/− SD RepeatsIC₅₀ +/− SD Repeats B 117 +/− 94 7 161 +/− 95  6 E 29 +/− 8 3 54 +/− 333 G  53 +/− 13 3 93 +/− 44 3 F  66 +/− 13 3 166 +/− 189 3 D 88 +/− 6 3110 +/− 14  3 C  97 +/− 31 3 186 +/− 194 3

d) IL-22 Assay

It is known that IL-23 is a potent inducer of proinflammatory cytokines.IL-23 acts on activated and memory T cells and promotes the survival andexpansion of Th17 cells which produce proinflammatory cytokinesincluding IL-22. In this assay, human whole blood is used to assess theability of the IL-23 antibodies to inhibit IL-23-induced IL-22production.

A whole blood assay is conducted in the same manner as described abovewith the modification of using rhuIL-23 or cynoIL-23 at 1 ng/ml andrhuIL-18 at 10 ng/ml to induce IL-22 production. IL-22 concentration isdetermined by IL-22 ELISA (R&D Systems, Minneapolis, Minn.).

As seen in TABLE 8, the antibodies potently inhibited rhuIL-23-inducedand cyno IL-23-induced IL-22 production in whole blood cells in a dosedependent manner. The antibodies all had IC₅₀ values in the picomolarrange.

TABLE 8 Table of mean IC₅₀ (pM) values for IL-23 antibodies in the IL-22human whole blood assay rhuIL-23 Cyno IL-23 antibody IC₅₀ +/− SD RepeatsIC₅₀ +/− SD Repeats B 117 +/− 68  4 113 +/− 65  3 E  87 +/− 109 3 56 +/−60 3 G 83 +/− 59 3 66 +/− 45 3

Example 3 Determining the Equilibrium Dissociation Constant (K_(D)) forAnti-IL-23 Antibodies Using KinExA Technology

Binding affinity of rhuIL-23 to IL-23 antibodies is evaluated using akinetic exclusion assay (KinExA assay, Sapidyne Instruments, Inc.,Boise, Id.). Normal human serum (NHS)-activated Sepharose 4 fast flowbeads (Amersham Biosciences, part of GE Healthcare, Uppsala, Sweden),are pre-coated with rhuIL-23 and blocked with 1 m Tris buffer with 10mg/mL BSA. 50 pM of IL-23 antibody is incubated with rhuIL-23 (12 pointsof 1:2 dilutions starting from 800 pM) at room temperature for 72 hoursbefore it is run through the rhuIL-23-coated Sepharose beads. The amountof the bead-bound antibody was quantified by fluorescent (Cy5) labeledgoat anti-human-Fc antibody (Jackson Immuno Research, West Grove, Pa.).The binding signal is proportional to the amount of free antibody atequilibrium.

The dissociation equilibrium constant (K_(D)) and the association rate(K_(on)) are obtained from curve fitting using KinExA Pro software. Thedissociation rate (K_(off)) is derived from: K_(D)=K_(off)/K_(on)

As seen in TABLE 9, the antibodies have high affinity for binding tohuman IL-23. All had K_(D) values in the low to sub pM range.

TABLE 9 Table of K_(D) (pM), K_(on) (1/MS) and K_(off) (1/s) ratesAntibody KD (pM) Kon (1/MS) Koff (1/s) E 0.131 9.12E+05 1.4E−07 D 0.1261.72E+06 2.2E−07 B 3.99 1.17E+06 4.7E−06 C 2.56 1.36E+06 4.1E−06 F 2.625.69E+05 1.5E−06 L 1.08 3.34E+06 3.7E−06 G 2.00 4.00E+05 8.1E−07

Example 4 Structure Determination Using X-Ray Crystallography

One way to determine the structure of an antibody-antigen complex is byusing X-ray crystallography, see for example, Harlow and LaneAntibodies: A Laboratory Manual Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y. (1990), p. 23. The crystal structure of IL-23has been determined, (see Lupardus and Garcia, J Mol Biol, 2008, 382:931-941) and the crystal structure of an IL-23/Fab complex has beendisclosed, (see Beyer et al. J Mol Biol, 2008. 382(4): 942-55).Structural determination of IL-23 with Fab fragments of antibodiesclaimed herein was obtained using X-ray crystallography.

Protein for Crystallization

A recombinantly derived human IL-23 heterodimer was used for thecrystallization studies (see Beyer et al., supra). The sequence of thehuman p19 subunit comprised of residues 20-189 of SEQ ID NO: 145, thesignal sequence of SEQ ID NO:154 and a C-terminal 6-His tag SEQ IDNO:155. The sequence of the human p40 subunit was mutated fromasparagine to glutamine at position 222 of SEQ ID NO:147 in order toprevent glycosylation at this site (Beyer, et al., supra).

Fabs derived from Antibody B and Antibody E were expressed on an IgG1scaffold that incorporated a caspase cleavage site. The Fabs wereprocessed by means of protease cleavage.

Complex Formation and Crystallization

The IL-23-Antibody B Fab complex was made by mixing a 2× molar excess ofthe Antibody B Fab with the human heterodimeric IL-23 described above.The complex was purified by size exclusion chromatography to removeexcess Antibody B Fab and concentrated to −12 mg/ml for crystallization.The IL-23-Antibody B Fab complex crystallized in 0.1 M Hepes pH 7, 8%PEG 8000.

The IL-23-Antibody E Fab complex was made by mixing a 2× molar excess ofthe Antibody E Fab with the human heterodimeric IL-23 described above.The complex was methylated using a JBS Methylation Kit according tomanufacturer's instructions (Jena Bioscience, Jena, Germany). Thecomplex was then treated with PNGase to deglycosylate the protein.Following these treatments, the complex was purified by size exclusionchromatography to remove excess Antibody E Fab and concentrated to 13.5mg/ml for crystallization. The IL-23-Antibody E Fab complex crystallizedin 0.1 M Tris pH 8.5, 0.2 M magnesium chloride, 15% PEG 4000.

Data Collection and Structure Determination

IL-23-Antibody B Fab crystals grew in the P2₁ space group with unit celldimensions a=70.93, b=71.27, c=107.37 Å, β=104.98° and diffract to 2.0 Åresolution. The IL-23-Antibody B Fab structure was solved by molecularreplacement with the program MOLREP (CCP4, The CCP4 suite: programs forprotein crystallography. Acta Crystallogr D Biol Crystallogr, 1994.50(Pt 5): p. 760-3) using the IL-23 structure (Beyer et al. supra) asthe starting search model. Keeping the IL-23 solution fixed, an antibodyvariable domain was used as a search model. Keeping the IL-23-antibodyvariable domain solution fixed, an antibody constant domain was used asa search model. The complete structure was improved with multiple roundsof model building with Quanta and refinement with cnx (Brunger, et al.,Acta Crystallogr D Biol Crystallogr, 1998, 54(Pt 5): p. 905-21).

Distances between protein atoms were calculated using the program PyMOL(DeLano, W. L. The PyMOL Graphics System. Palo Alto, 2002) (Schrodinger,LLC; New York, N.Y.)). Amino acids were chosen if at least one atom waslocated within the required distance threshold to the partner protein.

Boundaries of the A, B, C and D helices of the p19 subunit of IL-23 whenbound to the Antibody B Fab include A helix residues 28-47, B helixresidues 86-105, C helix residues 119-134 and D helix residues 154-187of SEQ ID NO:145.

The regions of interaction on the IL-23p19 subunit when bound to theAntibody B Fab include residues within Ser46-Glu58, Glu112-Glu123 andPro155-Phe163 of SEQ ID NO:145.

IL-23p19 subunit amino acid residues with atoms 4 Å or less from theAntibody B Fab include Ser46, Ala47, His48, Pro49, Leu50, His53, Met54,Asp55, Glu58, Pro113, Ser114, Leu115, Leu116, Pro120, Val121, Trp156,Leu159, Leu160, Arg162 and Phe163 of SEQ ID NO:145. IL-23p19 amino acidresidues with atoms between 4 Å and 5 Å from the Antibody B Fab includeVal51, Arg57, Glu112, Asp118, Ser119, Gln123, Pro155 of SEQ ID NO:145.

IL-23p40 subunit amino acid residues with atoms 4 Å or less from theAntibody B Fab include Glu 122 and Lys 124 of SEQ ID NO:147.

The Antibody B Fab heavy chain amino acid residues with atoms 4 Å orless from the IL-23 heterodimer include Gly32, Gly33, Tyr34, Tyr35,His54, Asn58, Thr59, Tyr60, Lys66, Arg101, Gly102, Phe103, Tyr104 andTyr105 of SEQ ID NO:46. The Antibody B Fab heavy chain amino acidresidues with atoms ≤5 Å from the IL-23 heterodimer include Ser31,Gly32, Gly33, Tyr34, Tyr35, His54, Ser56, Asn58, Thr59, Tyr60, Lys66,Arg101, Gly102, Phe103, Tyr104 and Tyr105 of SEQ ID NO:46.

The Antibody B Fab light chain amino acid residues with atoms 4 Å orless from the IL-23 heterodimer include Ser30, Ser31, Trp32, Tyr49,Ser52, Ser53, Ala91, Asn92, Ser93, Phe94, and Phe96 of SEQ ID NO:15. TheAntibody B Fab light chain amino acid residues with atoms ≤5 Å from theIL-23 heterodimer include Ser30, Ser31, Trp32, Tyr49, Ala50, Ser52,Ser53, Ser56, Ala91, Asn92, Ser93, Phe94, and Phe96 of SEQ ID NO:15

The IL-23-Antibody E Fab complex crystals grew in the P222₁ space groupwith unit cell dimensions a=61.60, b=97.59, c=223.95 Å and diffract to3.5 Å resolution. The IL-23-Antibody E Fab complex structure was solvedby molecular replacement with the program Phaser (CCP4, supra) using theIL-23 structure, an antibody variable domain, and an antibody constantdomain as the three starting search models, as described above. Thecomplete structure was improved with multiple rounds of model buildingwith Quanta and refinement with cnx (Brunger, et al., supra). TheAntibody E Fab constant domain was left out of the final refinedstructure due to very poor electron density for that portion of theprotein.

The regions of interaction on the IL-23p19 subunit identified when boundto the Antibody E Fab include residues within Ser46-His53, Glu112-Val120and Trp156-Phe163 of SEQ ID NO:145.

IL-23p19 amino acid residues with atoms 4 Å or less from the Antibody EFab include Ser46, Ala47, His48, Pro49, Leu50, Glu112, Pro113, Ser114,Leu115, Leu116, Pro117, Asp118, Ser119, Pro120, Trp156, Leu159, Leu160and Phe163 of SEQ ID NO: 145. IL-23p19 amino acid residues with atomsbetween 4 Å and 5 Å from the Antibody E Fab include His53 of SEQ IDNO:145.

IL-23p40 amino acid residues with atoms 4 Å or less from the Antibody EFab include Lys121, Glu 122, Pro123 and Asn 125 of SEQ ID NO:147.

The Antibody E Fab heavy chain amino acid residues with atoms 4 Å orless from the IL-23 heterodimer include Gly26, Phe27, Thr28, Ser31,Tyr53, Tyr59, Tyr102, Ser104, Ser105, Trp106, Tyr107, and Pro108 of SEQID NO:31. The Antibody E Fab heavy chain amino acid residues with atoms≤5 Å from the IL-23 heterodimer include Gln1, Gly26, Phe27, Thr28,Ser30, Ser31, Tyr32, Trp52, Tyr53, Tyr59, Arg100, Tyr102, Thr103,Ser104, Ser105, Trp106, Tyr107, and Pro108 of SEQ ID NO:31.

The Antibody E Fab light chain amino acid residues with atoms 4 Å orless from the IL-23 heterodimer include Ala31, Gly32, Tyr33, Asp34,Tyr51, Gly52, Asn55, Lys68, and Tyr93 of SEQ ID NO:1. The Antibody B Fablight chain amino acid residues with atoms ≤5 Å from the IL-23heterodimer include Thr29, Ala31, Gly32, Tyr33, Asp34, Tyr51, Gly52,Asn55, Lys68, Tyr93, and Trp100 of SEQ ID NO:1.

Example 5 Determination of IL-23-Antibody Complex Contact ResiduesThrough Solvent Accessible Surface Area Differences

The residue contacts in the paratope (the portion of the antibody thatrecognizes the antigen) and the portion of the antigen that it bindsbound by the paratope in a human IL-23-Antibody B Fab complex and in ahuman IL-23-Antibody E Fab complex were determined using solventaccessible surface area differences. The solvent accessible surface areacalculations were performed using Molecular Operating Environment(Chemical Computing Group, Montreal, Quebec).

The solvent accessible surface area differences of the paratope residuesin the IL-23-Antibody B Fab complex were calculated by setting theAntibody B Fab residues as the desired set. The structural informationobtained in Example 4 for the IL-23-Antibody B Fab complex was used andthe residue solvent accessible surface area of the amino acid residuesof the Antibody B Fab in the presence of the IL-23 heterodimer werecalculated and represent the “bound areas” for the set.

The residue solvent accessible surface area of each of the Antibody BFab residues in the absence of the IL-23 antigen were calculated andrepresent the “free areas” of the set.

The “bound areas” were then subtracted from the “free areas” resultingin the “solvent exposed surface area difference” for each residue in theset. The Antibody B Fab residues that had no change in surface area, ora zero difference, had no contact with the residues of the IL-23 antigenwhen complexed. The Antibody B Fab residues that had a differencevalue≥10 Å² were considered to be in significant contact with residuesin the IL-23 antigen such that these Antibody B Fab residues were atleast partially to completely occluded when the Antibody B Fab was boundto human IL-23. This set of Antibody B Fab residues make up the “coveredpatch”, the residues involved in the structure of the interface whenAntibody B Fab is bound to human IL-23, see Tables 10 and 11. TheAntibody B Fab residues in this covered patch may not be involved inbinding interactions with residues of the IL-23 antigen, but mutation ofany single residue within the covered patch could introduce energeticdifferences that would impact the binding of Antibody B Fab to humanIL-23. With the exception of Tyr49, all of the residues are located inthe CDR regions of the Antibody B Fab light and heavy chains. Theseresidues were also within 5 Å or less of the 11-23 antigen when bound tothe Antibody B Fab, as described in Example 4.

TABLE 10 Solvent Accessibility Surface Area Differences for Antibody BFab Light Chain Residue Residue Position Solvent exposed surface areaAHO Number SEQ ID NO: 15 difference (Å²) Ser32 Ser30 44.9 Ser33 Ser3141.1 Trp40 Trp32 79.0 Tyr57 Tyr49 40.7 Ala58 Ala50 20.3 Ser68 Ser52 43.6Ser69 Ser53 38.9 Ser72 Ser56 19.1 Asn110 Asn92 34.0 Phe135 Phe94 51.4

TABLE 11 Solvent Accessibility Surface Area Differences for Antibody BFab Heavy Chain Residue Residue Positioin Solvent exposed surface areaAHO Number SEQ ID NO: 46 difference (Å²) Ser33 Ser31 18.2 Gly34 Gly3249.5 Gly38 Gly33 33.8 Tyr39 Tyr34 51.4 Tyr40 Tyr35 30.7 His59 His54 29.5Asn67 Asn58 66.7 Thr68 Thr59 26.0 Tyr69 Tyr60 59.4 Lys75 Lys66 32.6Arg110 Arg101 47.2 Gly111 Gly102 21.7 Phe112 Phe103 35.5 Tyr133 Tyr10483.0 Tyr134 Tyr105 91.7

The solvent accessible surface area differences of the residues in theIL-23-Antibody E Fab complex were calculated as described above. TheAntibody E Fab residues that had a difference value≥10 Å² wereconsidered to be in significant contact with residues in the IL-23antigen and these Antibody E Fab residues were at least partially tocompletely occluded when the Antibody E Fab was bound to human IL-23.This set of Antibody E Fab residues make up the covered patch, theresidues involved in the structure of the interface when the Antibody EFab is bound to human IL-23, see Tables 12 and 13. The Antibody E Fabresidues in this covered patch may not be involved in bindinginteractions with residues of the IL-23 antigen, but mutation of anysingle residue within the covered patch could introduce energeticdifferences that would impact the binding of Antibody E Fab to humanIL-23. For the most part, these covered patch residues were locatedwithin the CDR regions of the Antibody E Fab heavy and light chains.These residues were also within 5 Å or less of the IL-23 antigen whenbound to the Antibody E Fab, as described in Example 4.

TABLE 12 Solvent Accessibility Surface Area Differences for Antibody EFab Light Chain Residue Residue Position Solvent exposed surface areaAHO Number SEQ ID NO: 1 difference (Å²) Ala33 Ala31 11.6 Gly34 Gly3251.2 Tyr39 Tyr33 47.2 Asp40 Asp34 36.8 Tyr57 Tyr51 16.1 Gly58 Gly52 11.1Asn69 Asn55 29.4 Lys82 Lys68 20.1 Tyr109 Tyr93 27.3 Ser135 Ser98 11.3

TABLE 13 Solvent Accessibility Surface Area Differences for Antibody EFab Heavy Chain Residue Residue Position Solvent exposed surface areaAHO Number SEQ ID NO: 31 difference (Å²) Gln1 Gln1 41.1 Gly27 Gly26 24.6Thr30 Thr28 82.2 Ser33 Ser31 40.7 Tyr39 Tyr32 30.7 Trp59 Trp52 11.3Tyr60 Tyr53 44.7 Tyr69 Tyr59 42.4 Lys86 Lys76 17.4 Gly111 Gly101 12.8Tyr112 Tyr102 103.1 Ser114 Ser104 21.0 Ser115 Ser105 91.4 Trp131 Trp106145.0 Tyr132 Tyr107 71.6 Pro133 Pro108 20.4

The solvent accessible surface area differences of the portion of theIL-23 heterodimer bound by the paratope of the Antibody B Fab werecalculated by setting the IL-23 heterodimer residues as the desired set.The structural information obtained in Example 4 for the Antibody BFab-IL-23 complex was used and the residue solvent accessible surfacearea of the amino acid residues of the IL-23 heterodimer in the presenceof the Antibody B Fab were calculated and represent the bound areas forthe set.

The residue solvent accessible surface area of each of the IL-23heterodimer residues in the absence of the Antibody B Fab werecalculated and represent the free areas of the set.

As described above, the bound areas were subtracted from the free areasresulting in the solvent exposed surface area difference for each IL-23residue. The IL-23 heterodimer residues that had no change in surfacearea, or a zero difference, had no contact with the residues of theAntibody B Fab when complexed. The IL-23 heterodimer residues that had adifference value≥10 Å² were considered to be in significant contact withresidues of the Antibody B Fab and these 11-23 heterodimer residues wereat least partially to completely occluded when the human IL-23heterodimer was bound to the Antibody B Fab. This set of IL-23heterodimer residues make up the covered patch, the residues involved inthe structure of the interface when the human IL-23 heterodimer is boundto the Antibody E Fab, see Table 14. The 11-23 heterodimer residues inthis covered patch may not all be involved in binding interactions withresidues on the Antibody B Fab, but mutation of any single residuewithin the covered patch could introduce energetic differences thatwould impact the binding of Antibody B Fab to human IL-23. Theseresidues are also within 4 Å or less from the Antibody B Fab, asdescribed Example 4.

TABLE 14 Solvent Accessibility Surface Area Differences for IL-23heterodimer residues Solvent exposed surface area difference (Å²) p19residues (SEQ ID NO: 145) Ser46 26.5 Ala47 12.7 Pro49 59.6 Leu50 122.2His53 47.8 Met54 13.9 Asp55 20.5 Arg57 14.6 Glu58 96.5 Glu112 29.7Pro113 64.8 Ser114 30.0 Leu115 31.4 Leu116 60.0 Asp118 14.4 Ser119 19.7Pro120 64.7 Pro155 19.4 Typ156 61.9 Leu159 72.8 Leu160 27.0 Arg162 14.4Phe163 67.5 p40 residues (SEQ ID NO: 147) Glu122 29.1 Lys124 60.9

The solvent accessible surface area differences of the portion of theIL-23 heterodimer bound by the paratope of the Antibody E Fab werecalculated as described above. The IL-23 heterodimer residues that had adifference value≥10 Å² were considered to be in significant contact withresidues of the Antibody E Fab and these 11-23 heterodimer residues wereat least partially to completely occluded when the human IL-23heterodimer was bound to the Antibody E Fab. This set of IL-23heterodimer residues make up the covered patch, the residues involved inthe structure of the interface when the human IL-23 heterodimer is boundto the Antibody E Fab, see Table 15. The 11-23 heterodimer residues inthis covered patch may not all be involved in binding interactions withresidues on the Antibody E Fab, but mutation of any single residuewithin the covered patch could introduce energetic differences thatwould impact the binding of Antibody E Fab to human IL-23. Theseresidues are also within 5 Å or less from the Antibody E Fab, asdescribed in Example 4.

TABLE 15 Solvent Accessibility Surface Area Differences for IL-23heterodimer residues Solvent exposed surface area difference (Å²) p19residues (SEQ ID NO: 145) Ser46 18.7 Ala47 14.9 Pro49 79.8 Leu50 99.5His53 61.2 Glu112 62.8 Pro113 45.7 Ser114 69.5 Leu115 50.3 Leu116 127.2Pro117 54.1 Asp118 37.0 Pro120 18.8 Pro155 16.9 Trp156 140.7 Leu159 21.8Leu160 17.0 Phe163 56.6 p40 residues (SEQ ID NO: 147) Lys121 86.2 Glu12221.8 Pro123 22.1 Asn125 26.7 Arg283 22.6

What is claimed is:
 1. An isolated nucleic acid molecule encoding one orboth variable regions of an antigen binding protein that binds IL-23,wherein the antigen binding protein comprises at least one heavy chainvariable region comprising: a CDRH1 of SEQ ID NO: 91, a CDRH2 of SEQ IDNO:92, and a CDRH3 of SEQ ID NO: 93; and at least one light chainvariable region comprising: a CDRL1 of SEQ ID NO: 62, a CDRL2 of SEQ IDNO:63, and a CDRL3 of SEQ ID NO:64.
 2. An isolated nucleic acid moleculeencoding one or both variable regions of an isolated antigen bindingprotein that binds IL-23, wherein the antigen binding protein comprisesa heavy chain variable region comprising amino acid residues 31-35,50-65 and 99-113 of SEQ ID NO:31; and a light chain variable regioncomprising amino acid residues 23-36, 52-58 and 91-101 of SEQ ID NO:1.3. An isolated nucleic acid molecule encoding one or both variableregions of an isolated antigen binding protein that binds IL-23, whereinthe antigen binding protein comprises a heavy chain variable region ofSEQ ID NO: 31 and a light chain variable region of SEQ ID NO:
 1. 4. Theisolated nucleic acid molecule of claim 3, wherein the nucleic acidencoding the heavy chain variable region is SEQ ID NO:32.
 5. Theisolated nucleic acid molecule of claim 3, wherein the nucleic acidencoding the light chain variable region is SEQ ID NO:2.
 6. The isolatednucleic acid molecule of claim 3, wherein the nucleic acid encoding theheavy chain variable region is SEQ ID NO:32 and the nucleic acidencoding the light chain variable region is SEQ ID NO:2.
 7. A nucleicacid molecule according to claim 1, 2 or 3, wherein said nucleic acidmolecule is operably linked to a control sequence.
 8. A nucleic acidmolecule according to claim 6, wherein said nucleic acid molecule isoperably linked to a control sequence.
 9. An isolated vector comprisinga nucleic acid molecule according to claim 1, 2 or
 3. 10. An isolatedvector comprising a nucleic acid molecule according to claim
 6. 11. Anisolated host cell comprising the nucleic acid molecule according toclaim 1, 2 or
 3. 12. An isolated host cell comprising the nucleic acidmolecule according to claim
 6. 13. An isolated host cell comprising thevector according to claim
 9. 14. An isolated host cell comprising thevector according to claim
 10. 15. A method of making an antigen bindingprotein that binds IL-23 comprising the step of collecting said antigenbinding protein from a host cell of claim 11, wherein the host cellcomprises nucleic acid encoding both the heavy chain variable region andthe light chain variable region of the antigen binding protein.
 16. Amethod of making an antigen binding protein that binds IL-23 comprisingthe step of collecting said antigen binding protein from a host cell ofclaim 12, wherein the host cell comprises nucleic acid encoding both theheavy chain variable region and the light chain variable region of theantigen binding protein.
 17. A method of making an antigen bindingprotein that binds IL-23 comprising the step of collecting said antigenbinding protein from a host cell of claim 13, wherein the host cellcomprises nucleic acid encoding both the heavy chain variable region andthe light chain variable region of the antigen binding protein.
 18. Amethod of making an antigen binding protein that binds IL-23 comprisingthe step of collecting said antigen binding protein from a host cell ofclaim 14, wherein the host cell comprises nucleic acid encoding both theheavy chain variable region and the light chain variable region of theantigen binding protein.